RU2796937C2 - Monoclonal antibody or its antigen-binding fragment which specifically binds to gd2 (gd2 ganglioside) and its use - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: group of inventions relates namely to a monoclonal antibody or its antigen-binding fragment, which specifically binds to GD2 (GD2 ganglioside). The invention also relates to nucleic acids encoding the said antibody, expression vectors, host cells and methods for their production, methods for producing antibodies of the invention, pharmaceutical compositions containing an antibody of the invention, pharmaceutical compositions containing an antibody of the invention and other therapeutically active compounds, methods treatment of GD2 mediated diseases or disorders, uses of the antibodies or pharmaceutical compositions thereof for the treatment of GD2 mediated diseases or disorders, and uses of the antibodies of the invention and other therapeutically active compounds for the treatment of GD2 mediated diseases or disorders.

EFFECT: invention makes it possible to obtain new antibodies with high affinity for HLA-G.

73 cl, 6 dwg, 11 tbl, 9 ex

Description

The field of technology to which the invention belongs

The present invention relates to the field of biotechnology and medicine, namely to a monoclonal antibody or antigen-binding fragment that specifically binds to GD2 (ganglioside GD2). The invention also relates to nucleic acids encoding said antibody, expression vectors, host cells and methods for their production, methods for producing antibodies according to the invention, pharmaceutical compositions containing an antibody according to the invention, pharmaceutical compositions containing an antibody according to the invention and other therapeutically active compounds, methods treatment of GD2 mediated diseases or disorders, uses of the antibodies or pharmaceutical compositions thereof for the treatment of GD2 mediated diseases or disorders, and uses of the antibodies of the invention and other therapeutically active compounds for the treatment of GD2 mediated diseases or disorders.

State of the art

GD2 is the first ganglioside to be an effective target antigen for cancer immunotherapy.

Gangliosides are composed of glycosphingolipids and sialic acids (N-acetylneuraminic acid, Neu5Ac or NANA), which are monosaccharides with nine carbon atoms. Ganglioside nomenclature is based on the number and position of NANA residues. Monosaccharides are first added to ceramide to form lactosylceramide, and then NANA residues are added to form gangliosides. Each sugar is linked by specific glycosyltransferases. GD2 has two NANAs (a-2,8-sialic acid and a-2,3-sialic acid) and is derived from the precursor GD3 by addition of Ga1-NAc via the enzyme GM2/GD2 synthase (b1,4-N-acetylgalactosaminyltransferase). The terminal pentaoligosaccharide constitutes the specific GD2 epitope to which the most specific antibodies are directed. This critical OM2/OB2 synthase enzyme responsible for GD2 formation has been successfully used as a molecular marker for minimal residual neuroblastoma in the bone marrow, with a strong predictive effect on patient survival. As shown in ganglioside synthesis pathways, epitope proximity for GD2 can be clearly defined. For example, GD3 and GD1b are the most common cross-reactive gangliosides recognized by anti-GD2 antibodies. A derivative of GD2 with a 9-O-acetyl modification at the terminal sialic acid is called O-acetyl-CD2. While most anti-GD2 antibodies cross-react with O-GD2, some do not. Anti-O-GD2 antibodies without cross-reactivity with GD2 had less cross-reactivity with normal neurons. Gangliosides are found on the surface of cells of the nervous system of vertebrates. Lower vertebrates, such as fish and amphibians, have more polysialo-gangliosides containing four to five NANA residues, while gangliosides of higher vertebrates, including reptiles, birds, and mammals, have only one to three NANA residues (MAYA SUZUKI ET AL ., Disialoganglioside GD2 as a therapeutic target for human diseases, Expert Opinion on Therapeutic Targets, 2015, v. 15, pages 349-362, PMID: 25604432, DOI 10.1517/14728222.2014.986459).

GD2 is expressed on neural stem cells, mesenchymal stem cells (MSCs), and peripheral sympathoadrenergic progenitors and is involved in neuronal differentiation and proliferation. While the role of polysialic acid in neuronal development has been extensively studied, the exact functions of gangliosides, and GD2 in particular, remain unknown. After birth, GD2 expression is restricted to the CNS, predominantly by neuronal cell bodies and MSCs, as well as peripheral nerves and skin melanocytes at low levels. GD2 is believed to play a role in the maintenance and repair of neural tissues that undergo progressive degenerative changes through the regulation of complement activation and subsequent inflammation, although the exact immunological mechanism remains unclear. It should be noted that GD2 (+) MSCs have the potential to differentiate into several clones, including neurons (MAYA SUZUKI ET AL., Disialoganglioside GD2 as a therapeutic target for human diseases, Expert Opinion on Therapeutic Targets, 2015, v. 15, pages 349- 362, PMID: 25604432, DOI: 10.1517/14728222.2014.986459).

GD2 is overexpressed in various embryonic cancers (neuroblastoma, brain tumors, retinoblastoma, Ewing's sarcoma, rhabdomyosarcoma), bone tumors (osteosarcoma, Ewing's sarcoma), soft tissue sarcomas (leiomyosarcoma, liposarcoma, fibrorosarcoma), lung cancer, melanoma, and breast cancer.

Tumor monoclonal antibodies (mAbs) have demonstrated efficacy in the clinic, becoming an important method of cancer immunotherapy. Due to its limited expression in normal tissue, GD2 disialogloside expressed on neuroblastoma cells is an excellent candidate for mAb therapy.

WO2005070967A2 describes an anti-GD2 antibody.

Currently, only 2 antibodies to GD2 are approved for therapeutic use in the world (dinutuximab and nataximab). In connection with the foregoing, it is relevant to create new antagonistic antibodies that specifically bind to GD2.

Description of the invention

Brief description of the invention

The authors of this group of inventions have developed antibodies that specifically bind to GD2, and also have at least 80 percent degree of humanization in variable domains. These antibodies have high thermal stability.

In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 (GD2 ganglioside), wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable domain comprising:

(i) a CDR1 with an amino acid sequence selected from the group: GHNMN (SEQ ID NO: 1) or GKNMN (SEQ ID NO: 2),

(ii) CDR2 with amino acid sequence

(iii) a CDR3 with an amino acid sequence selected from the group: GMIY (SEQ ID NO: 4), GMFY (SEQ ID NO: 5), GMYY (SEQ ID NO: 6) or GMLY (SEQ ID NO: 7); And

(b) a light chain variable domain comprising:

(i) a CDR1 with an amino acid sequence selected from the group: RSSRLVHRNGNTYLH (SEQ ID NO: 8) or

(ii) a CDR2 with an amino acid sequence selected from the group: KVSNRFG (SEQ ID NO: 10) or KVNNRFS (SEQ ID NO: 11),

(iii) a CDR3 with an amino acid sequence selected from the group: GQSTHVPPLT (SEQ ID NO: 12) or SQSTHVPPLS (SEQ ID NO: 13).

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with amino acid sequence

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 45).

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with an amino acid sequence selected from the group:

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with the amino acid sequence FGQGTKLELK (SEQ ID NO: 50).

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

a) a heavy chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with amino acid sequence

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 45), and where

b) a light chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with an amino acid sequence selected from the group:

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with amino acid sequence

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 9,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 11,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 10,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains an amino acid sequence that has at least 98 percent identity with the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains an amino acid sequence that has at least 96 percent identity with the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains an amino acid sequence that has at least 98 percent identity with the amino acid sequence of SEQ ID NO: 17;

(b) a light chain variable domain that contains an amino acid sequence that has at least 96 percent identity with the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17;

(b) a light chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains the amino acid sequence of SEQ ID NO: 16;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains the amino acid sequence of SEQ ID NO: 17;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody that specifically binds to GD2 is a full length IgG antibody.

In some embodiments, the isolated monoclonal antibody is a full-length IgG antibody that is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the isolated monoclonal antibody is a full length IgG antibody that is of the human IgG1 isotype.

In some embodiments, the isolated monoclonal antibody contains YTE (M252Y, S254T, T256E) and/or K322A mutations in the Fc fragment compared to the natural sequence of the Fc fragment.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 , SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37.

In some embodiments, the isolated monoclonal antibody comprises a light chain containing an amino acid sequence selected from the group: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

In some embodiments, the isolated monoclonal antibody comprises:

(a) a heavy chain which contains an amino acid sequence selected from the group: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, and

(b) a light chain that contains an amino acid sequence selected from the group: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

In some embodiments, the isolated monoclonal antibody comprises:

(a) a heavy chain which contains the amino acid sequence of SEQ ID NO: 32; And

(b) a light chain that contains the containing amino acid sequence of SEQ ID NO: 39.

In some embodiments, the isolated monoclonal antibody comprises:

(a) a heavy chain which contains the amino acid sequence of SEQ ID NO: 33; And

(b) a light chain that contains the amino acid sequence of SEQ ID NO: 41.

In one aspect, the present invention relates to an isolated nucleic acid that encodes any of the above antibody or antigennegative fragment.

In some embodiments, the isolated nucleic acid is DNA.

In one aspect, the present invention relates to an expression vector that contains any of the above nucleic acids.

In one aspect, the present invention relates to a method for obtaining a host cell for obtaining any of the above antibodies or antigen-binding fragment and includes transforming the cell with the above vector.

In one aspect, the present invention relates to a host cell for obtaining any of the above antibodies or antigennegative fragment, which contains any of the above nucleic acids.

In one aspect, the present invention relates to a method for producing any of the above antibodies or antigen-binding fragment, which consists in culturing the above host cell in a culture medium under conditions sufficient to obtain the specified antibody, if necessary, followed by isolation and purification of the resulting antibody.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by GD2, which contains any of the above antibody or antigennegative fragment in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

In some embodiments of the pharmaceutical composition, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma , Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by GD2, which contains any of the above antibody or antigennegative fragment and at least one other therapeutically active compound.

In some embodiments of the pharmaceutical composition, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma , Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an antibody, a chemotherapeutic agent, or a hormone therapy agent.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an immune checkpoint inhibitor.

In some embodiments of the pharmaceutical composition, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In some embodiments of the pharmaceutical composition, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments of the pharmaceutical composition, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the pharmaceutical composition, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments of the pharmaceutical composition, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab.

In some embodiments of the pharmaceutical composition, the PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

In some embodiments of the pharmaceutical composition, the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

In one aspect, the present invention provides a method for inhibiting GD2 biological activity in a subject in need of such inhibition, which comprises administering to the subject an effective amount of any of the foregoing antibodies or antigen-binding fragment thereof.

In one aspect, the present invention relates to a method of treating a disease or disorder mediated by GD2, comprising administering to a subject in need of such treatment, any of the above antibodies or antigen-binding fragment or the above pharmaceutical composition in a therapeutically effective amount.

In one aspect, the present invention relates to a method of treating a disease or disorder mediated by GD2, comprising administering to a subject in need of such treatment, any of the above antibodies or antigen-binding fragment and selected from the group:

a) administering at least one other therapeutically active compound,

b) radiation therapy,

c) transplantation of hematopoietic stem cells,

d) surgical treatment and, if necessary, adjuvant therapy,

or

e) any combination of the above a)-d).

In some embodiments of the method of treatment, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma , Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

In some embodiments of the method of treatment, the other therapeutically active compound is an antibody, a chemotherapeutic agent, or a hormone therapy agent.

In some embodiments of the method of treatment, the other therapeutically active compound is an immune checkpoint inhibitor.

In some embodiments of the method of treatment, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In some embodiments of the method of treatment, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments of the method of treatment, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the method of treatment, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments of the method of treatment, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab.

In some embodiments of the method of treatment, the PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

In some embodiments of the treatment method, the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the method of treatment, the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

In one aspect, the present invention relates to the use of the above antibody or antigen-binding fragment or the above pharmaceutical composition for the treatment of a GD2 mediated disease or disorder in a subject in need of such treatment.

In one aspect, the present invention relates to the use of any of the above antibodies or antigen-binding fragment and at least one of the group:

a) another therapeutically active compound,

b) radiation therapy,

c) transplantation of hematopoietic stem cells or

d) surgical treatment and, if necessary, adjuvant therapy, for the treatment of a disease or disorder mediated by GD2.

In some embodiments, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

In some embodiments, the other therapeutically active compound is an immune checkpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab.

In some embodiments, the PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

In some embodiments, the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments, the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

Brief description of the drawings

Figure 1 is the structure of the Fab fragment in complex with the GD2 target.

Figure 2 is a map of the vector carrying the anni-GD2 antibody heavy chain genetic sequence.

Figure 3 is a map of the vector carrying the genetic sequence of the anra-GD2 antibody light chain. For figures 2-3

Figure 4 is an electropherogram of candidates 10-008 under reducing and non-reducing conditions, 4-20% SDS-PAGE gradient gel.

1. Molecular weight marker;

2. 10-008 1 µg under non-reducing conditions;

3. 10-008 1 µg under reducing conditions.

Figure 5 is a graph showing the presence of antibody-dependent cellular cytotoxicity of antibody 10-008 in the SK-N-BE(2) target cell assay. Points without antibody addition were used as a negative control.

Figure 6 is a graph showing the complement-dependent cytotoxicity of anti-GD2 antibodies of the invention in the SK-N-BE(2) target cell assay. The graph shows the dependence of the level of fluorescence of the vital dye (reflects the number of living cells) on the concentration of antibodies when adding human serum. In the SigmaPlot 14.0 program, EC50 values were obtained based on the logistic model.

Definitions and Common Methods

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have meanings that are commonly understood by those skilled in the art.

In addition, unless the context requires otherwise, terms in the singular include terms in the plural, and terms in the plural include terms in the singular. The generally used classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and widely used in this area. Enzymatic reactions and purification methods are carried out according to the manufacturer's instructions, as is commonly done in the art, or as described herein.

The term "Ka" as used herein refers to the rate of association of a particular antibody-antigen interaction, while the term "KD" or "Kd" refers to the rate of dissociation of a particular antibody-antigen interaction.

"Binding affinity" generally refers to the strength of the cumulative non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, "binding affinity" refers to intrinsic (intrinsic, intrinsic) binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by a dissociation constant (Kd). Desirably, the Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. Affinity can be measured by conventional methods known in the art, including the methods described herein. Low affinity antibodies usually bind antigen slowly and tend to dissociate easily, while high affinity antibodies usually bind antigen faster and tend to stay bound longer. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.

The term "koff" or "kdis" refers to the dissociation rate constant of a particular interaction between a binding molecule and an antigen. The dissociation rate constant koff can be measured by biolayer interferometry, for example using the Octet™ system.

The term "kon" or "on-rate" refers to the association rate constant.

The term "off-rate screening" refers to a screening in which candidates are screened based on koff values alone.

The term "R ² " refers to the coefficient of determination.

The term "Response" refers to the binding signal of an antibody to an antigen.

The term "in vitro" refers to a biological entity, biological process or biological response outside the body, simulated in vitro. For example, in vitro cell growth should be understood as the growth of cells in an environment outside the body, such as in a test tube, culture flask or microplate.

The term "IC50" (50% inhibitory concentration) refers to drug concentrations at which a measurable activity or response, such as cell growth or proliferation, such as tumor cells, is inhibited by 50%. The IC50 value can be estimated using appropriate log-dose response curves using special curve processing statistical programs.

The term ED50 (EC50) (50% effective dose/concentration) refers to drug concentrations at which 50% of a measurable biological effect is achieved (may include cytotoxicity).

In the present description and in the following claims, unless the context otherwise requires, the words "have", "include" and "comprise" or variations thereof, such as "has", "having", "includes", "including", " contains" or "comprising" should be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers.

Detailed description of the invention

Antibody

The present invention relates to a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2 (ganglioside GD2).

The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated by a single clonal population of cells.

The antibody of the invention is a recombinant antibody.

The term "recombinant antibody" means an antibody that is expressed in a cell or cell line containing a nucleotide sequence(s) that encodes an antibody, said nucleotide sequence(s) not naturally associated with the cell.

In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 (GD2 ganglioside), wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable domain comprising:

(i) a CDR1 with an amino acid sequence selected from the group: GHNMN (SEQ ID NO: 1) or GKNMN (SEQ ID NO: 2),

(ii) CDR2 with amino acid sequence

(iii) a CDR3 with an amino acid sequence selected from the group:

6) or GMLY (SEQ ID NO: 7); And

(b) a light chain variable domain comprising:

или(i) a CDR1 with an amino acid sequence selected from the group:

or

(ii) a CDR2 with an amino acid sequence selected from the group: KVSNRFG (SEQ ID NO: 10) or KVNNRFS (SEQ ID NO: 11),

(iii) a CDR3 with an amino acid sequence selected from the group: GQSTHVPPLT (SEQ ID NO: 12) or SQSTHVPPLS (SEQ ID NO: 13).

The definition of "isolated" ("isolated"), used to describe the various antibodies according to this description, means an antibody identified and isolated and/or regenerated from the cell or cell culture in which it is expressed. Impurities (contaminants) from the natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Typically, an isolated polypeptide is obtained by at least one purification step.

Amplification of the GD2 gene and/or overexpression of its protein have been found in many cancers, for example, in any of the diseases from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

The term "antibody" or "immunoglobulin" (Ig) as used herein includes whole antibodies. The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Five types of heavy chains of mammalian antibodies are known, which are denoted by Greek letters: a, δ, ε, γ and μ. (Janeway C.A., Jr. et al., Immunobiology, 5th ed., Garland Publishing, 2001). The type of heavy chain present determines the class of the antibody; these chains are found in antibodies of the type IgA, IgD, IgE, IgG and IgM, respectively (Rhoades R.A., Pflanzer R.G., Himan Physiology, 4th ed., Thomson Learning, 2002). Various heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε consist of approximately 550 amino acids. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The heavy chains γ, α and β contain a constant region which consists of the three constant domains CH1, CH2 and CH3 (lined up) and a hinge region which confers flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, pp. 89-99); heavy chains μ and ε contain a constant region that consists of four constant domains CH1, CH2, CH3 and CH4 (Janeway C.A., Jr. et al., Immunobiology, 5th ed., Garland Publishing, 2001 ). In mammals, only two types of light chains are known, which are referred to as lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably the light chain is a kappa (κ) light chain and the CL constant domain is preferably C-kappa (κ).

"Antibodies" according to the invention may be antibodies of any class (e.g. IgA, IgD, IgE, IgG and IgM, preferably IgG) or subclass (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl).

The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), scattered between regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a P(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CHI domains; (iv) an Fv fragment consisting of VL and VH domains in a single antibody arm, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a VH/VHH domain. In addition, the two regions of the Fv fragment, VL and VH, are encoded by different genes, they can be connected using recombinant methods using a synthetic linker, which makes it possible to obtain them as a single protein chain, in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 -5883). It is contemplated that such single chain molecules are also included in the term "antigen binding portion" of an antibody. Such antibody fragments are obtained using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is unevenly distributed over the 110 amino acid variable domain region. In contrast, V regions are composed of invariant fragments called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" or CDRs. Each native heavy and light chain variable domain contains four FRs, generally assuming a beta-sheet configuration linked by three hypervariable regions that form binding loops and in some cases are part of a beta sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, M.D. (1991)). The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, a hypervariable region contains amino acid residues from a "complementarity determining region" or "CDR" and/or such residues from a "hypervariable loop".

"Kabat nomenclature" or "Kabat nomenclature" is used in this application to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than the rest of the amino acid residues in the heavy and light chain variable regions of an antibody (Kabat et al. Ann N.Y. Acad. Sci., 190:382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

An antibody of the invention that "binds" a target antigen is an antibody that binds an antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent when targeted to a protein or cell or tissue expressing the antigen, and in slightly cross-reacts with other proteins. According to analytical methods: fluorescence-activated cell sorting (FACS), radioimmunoprecipitation (RIA) or ELISA (ELISA), in such embodiments of the invention, the degree of binding of the antibody to a protein that is not a “target” (“non-target protein”) is less than 10 % of antibody binding to a specific target protein. In relation to the binding of an antibody to a target molecule, the term "specific binding" or the expressions "specifically binds to" or "specific to" a particular polypeptide or epitope on a particular target polypeptide means a binding that is markedly (measurably) different from a non-specific interaction.

Specific binding can be quantified, for example, by determining the binding of the molecule compared to the binding of a control molecule. For example, specific binding can be determined by a competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specific binding" or the expressions "specifically binds to" or "specific to" a particular polypeptide or epitope on a particular target polypeptide can be characterized by an example of a molecule having a Kd to the target of at least about 200 nM, or at least at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or higher. In one embodiment, the term "specific binding" refers to a binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with the amino acid sequence WVRQNIGQGLEWMG (SEQ ID NO: 43),

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 45).

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with an amino acid sequence selected from the group:

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with the amino acid sequence FGQGTKLELK (SEQ ID NO: 50).

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

a) a heavy chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with amino acid sequence

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 45), and where

b) a light chain variable domain that contains:

(i) FR1 with amino acid sequence

(ii) FR2 with an amino acid sequence selected from the group:

(iii) FR3 with amino acid sequence

и

And

(iv) FR4 with amino acid sequence

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 9,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 11,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 10,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains an amino acid sequence that has at least 98 percent identity with the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that contains an amino acid sequence selected from the group:

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains an amino acid sequence that has at least 96 percent identity with the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain that contains an amino acid sequence selected from the group:

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains an amino acid sequence that has at least 98 percent identity with the amino acid sequence of SEQ ID NO: 17;

(b) a light chain variable domain that contains an amino acid sequence that has at least 96 percent identity with the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17;

(b) a light chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains the amino acid sequence of SEQ ID NO: 16;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a heavy chain variable domain that contains the amino acid sequence of SEQ ID NO: 17;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the isolated monoclonal antibody that specifically binds to GD2 is a full length IgG antibody.

In some embodiments, the isolated monoclonal antibody is a full-length IgG antibody that is of the human IgGl, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the isolated monoclonal antibody is a full length IgG antibody that is of the human IgGl isotype.

In some embodiments, the isolated monoclonal antibody contains YTE (M252Y, S254T, T256E) and/or K322A mutations in the Fc fragment compared to the natural sequence of the Fc fragment.

The above mutations in the Fc fragment are numbered according to the amino acid numbering of EU antibody chains (Edelman G.M. et al., Proc. Natl. Acad. Sci. USA 63, 1969, ee. 78-85; Kabat E.A. et al., Sequences of Proteins of Immunological Interest , 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

In some embodiments, the isolated monoclonal antibody comprises a heavy chain containing an amino acid sequence selected from the group:

In some embodiments, the isolated monoclonal antibody comprises a light chain containing an amino acid sequence selected from the group:

In some embodiments, the isolated monoclonal antibody comprises:

(a) a heavy chain which contains an amino acid sequence selected from the group: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, and

(b) a light chain that contains an amino acid sequence selected from the group: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

In some embodiments, the isolated monoclonal antibody comprises:

(a) a heavy chain which contains the amino acid sequence of SEQ ID NO: 32; And

(b) a light chain that contains the containing amino acid sequence of SEQ ID NO: 39.

In some embodiments, the isolated monoclonal antibody comprises:

(a) a heavy chain that contains the amino acid sequence of SEQ ID NO: 33; And

(b) a light chain that contains the amino acid sequence of SEQ ID NO: 41.

In some embodiments, an isolated monoclonal antibody that specifically binds to GD2 is antibody 07-006.

Antibody 07-006 includes:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 22; And

(b) a light chain containing the amino acid sequence of SEQ ID NO: 38.

Antibody 07-006 includes:

(a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 14;

(b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 18.

Antibody 07-006 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 6, and

(b) a light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 10,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 12.

Antibody 07-006 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 51,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 53,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 56, and

(b) a light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 58,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 60,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 62.

Antibody 07-006 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 64,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 66,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 69, and

(b) a light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 71,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 73,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 75.

In some embodiments, an isolated monoclonal antibody that specifically binds to GD2 is antibody 07-015.

Antibody 07-015 includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 23; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-015 includes:

(a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 15;

(b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 19.

Antibody 07-015 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 7, and

(b) a light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 10,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 12.

Antibody 07-015 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 52,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 53,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 57, and

(b) a light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 58,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 60,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 62.

Antibody 07-015 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 65,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 66,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 70, and

(b) a light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 71,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 73,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 75.

In some embodiments, an isolated monoclonal antibody that specifically binds to GD2 is antibody 07-016.

Antibody 07-016 includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 23; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-016 includes:

(a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 15;

(b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 18.

Antibody 07-016 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 7, and

(b) a light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 10,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 12.

Antibody 07-016 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 52,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 53,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 57, and

(b) a light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 58,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 60,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 62.

Antibody 07-016 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 65,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 66,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 70, and

(b) a light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 71,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 73,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 75.

In some embodiments, an isolated monoclonal antibody that specifically binds to GD2 is antibody 07-028.

Antibody 07-028 includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 23; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 40.

Antibody 07-028 includes:

(a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 15;

(b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 20.

Antibody 07-028 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 7, and

(b) a light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 11,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 13.

Antibody 07-028 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 52,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 53,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 57, and

(b) a light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 58,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 61,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 63.

Antibody 07-028 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 65,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 66,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 70, and

(b) a light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 71,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 74,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 76.

In some embodiments, an isolated monoclonal antibody that specifically binds to GD2 is antibody 07-031.

Antibody 07-031 includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 24; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-031 includes:

(a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 16;

(b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 19. Antibody 07-031 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 4, and

(b) a light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 8,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 10,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 12.

Antibody 07-031 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 51,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 53,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 54, and

(b) a light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 58,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 60,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 62.

Antibody 07-031 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 64,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 66,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 67, and

(b) a light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 71,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 73,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 75.

In some embodiments, an isolated monoclonal antibody that specifically binds to GD2 is antibody 07-041.

Antibody 07-041 includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 25; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 41.

Antibody 07-041 includes:

(a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 17;

(b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 21.

Antibody 07-041 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 2,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 3,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 5, and

(b) a light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 9,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 11,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 12.

Antibody 07-041 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 51,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 53,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 55, and

(b) a light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 59,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 61,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 62.

Antibody 07-041 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 64,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 66,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 68, and

(b) a light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 72,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 74,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 75.

Proposed modification(s) amino acid sequences of antibodies. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Variants of the amino acid sequence of an antibody are obtained by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is carried out to produce the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also change post-translational processes in the antibody, such as changing the number or position of glycosylation sites.

Modification of the amino acid sequences of antibodies using amino acid substitutions. Such a variant is the replacement of at least one amino acid residue in the antibody molecule with another residue. The sites of greatest interest for substitution mutagenesis include the hypervariable regions or CDRs, but changes in the FR or Fc regions are also contemplated. Conservative substitutions are shown in Table 1 under the heading "preferred substitutions". If such substitutions result in a change in biological activity, then further substantive changes may be introduced, referred to as "examples of substitutions" in Table A, or changes further described below in the description of amino acid classes, and screening of products may be performed.

In some embodiments, antibodies 07-006,07-015, 07-016, 07-028, 07-031, 07-041 include an Fc fragment that contains the YTE (M252Y, S254T, T256E) and/or K322A mutations compared with the natural sequence of the Fc fragment.

In some embodiments, antibodies 07-006,07-015, 07-016, 07-028, 07-031, 07-041 comprise an Fc fragment that contains the YTE (M252Y, S254T, T256E) and K322A mutations compared to native the Fc sequence of the fragment.

In some embodiments, antibodies 07-006, 07-015, 07-016, 07-028, 07-031, 07-041 include an Fc fragment that contains YTE mutations (M252Y, S254T, T256E) compared to the natural Fc sequence fragment.

In some embodiments, antibodies 07-006, 07-015, 07-016, 07-028, 07-031, 07-041 comprise an Fc fragment that contains the K322A mutation compared to the natural sequence of the Fc fragment.

Antibody 07-006 with YTE and K322A mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 30; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-015 with YTE and K322A mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 31; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-016 with YTE and K322A mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 31; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-028 with YTE and K322A mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 31; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 40.

Antibody 07-031 with YTE and K322A mutations (or antibody 10-02) includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 32; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-041 with YTE and K322A mutations (or antibody 10-08) includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 33; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 41.

Antibody 07-006 with YTE mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 26; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-015 with YTE mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 27; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-016 with YTE mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 27; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-028 with YTE mutations includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 27; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 40.

Antibody 07-031 with YTE mutations (or antibody 10-01) includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 28; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-041 with YTE mutations (or antibody 10-07) includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 29; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 41.

Antibody 07-006 with the K322A mutation includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 34; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-015 with the K322A mutation includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 35; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-016 with the K322A mutation includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 35; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 38.

Antibody 07-028 with the K322A mutation includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 35; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 40.

Antibody 07-031 with the K322A mutation (or antibody 10-03) includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 36; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 39.

Antibody 07-041 with the K322A mutation (or antibody 10-09) includes:

(a) a heavy chain containing the amino acid sequence of SEQ IDNO: 37; And

(b) a light chain containing the amino acid sequence of SEQ IDNO: 41.

In some embodiments, the antibodies of the invention may be afucosylated.

In some embodiments, the antibodies of the invention may be fucosylated.

The presence or absence of antibody fucosylation will depend on the cell culture used to produce the antibodies of the invention.

Antibody fragments

In certain circumstances, it is appropriate to use antibody fragments rather than complete antibodies. The smaller size of the fragments facilitates their rapid clearance and may contribute to better penetration into solid tumors.

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments have been prepared by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods, 24, 1992, pp. 107-117 and Brennan et al., Science, 229, 1985, p. 81). However, at present, these fragments can be obtained directly using recombinant host cells. Fab, Fv and scFv antibody fragments can be expressed and secreted from E. coli to facilitate the production of large amounts of these fragments. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly isolated from E. coli and chemically linked to form F(ab')2 fragments (Carter et al., Bio/Technology 10, 1992, pp. 163-167). According to another approach, F(ab')2 fragments can be isolated directly from a culture of recombinant host cells. Fab and F(ab')2 fragments with increased in vivo half-life that retain epitope-binding receptor residues are described in US 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93/16185; US 5571894 and US US 5587458). Fv and scFv are the only species with intact binding sites lacking constant regions; as a result, they can be used for reduced non-specific binding when used in vivo. Fusion proteins carrying scFv can be engineered to produce an effector protein fusion at either the N- or C-terminus of the scFv (see Antibody Engineering, ed. Borrebaeck, supra). The antibody fragment may also be a "linear antibody", such as described in US 5641870.

nucleic acid molecule

In one aspect, the present invention provides an isolated nucleic acid that encodes any of the aforementioned antibodies, or an antigen-binding fragment thereof, that specifically binds to GD2.

In any of the above embodiments, the nucleic acid molecules may be isolated.

The terms "nucleic acid", "nucleic acid sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this specification, refer to a distinct sequence of nucleotides, whether or not modified. , defining a fragment or region of a nucleic acid, containing or not containing non-natural nucleotides and which is either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of these DNA.

It should also be mentioned here that the present invention does not apply to nucleotide sequences in their natural chromosomal environment, i.e. in a natural state. The sequences of the present invention have been isolated and/or purified, i. were taken directly or indirectly, for example, by copying, while their environment was at least partially modified. Thus, isolated nucleic acids obtained by genetic recombination, for example by means of host cells, or obtained by chemical synthesis, are also to be understood here.

Reference to the nucleotide sequence covers his compliment unless otherwise noted. Thus, reference to a nucleic acid having a particular sequence is to be understood as encompassing its complementary strand with its complementary sequence.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of an antibody nucleic acid. An isolated nucleic acid molecule is different from the form or set in which it occurs naturally. Thus, the isolated nucleic acid molecule is different from the nucleic acid molecule that exists naturally in cells.

In one aspect, the present invention relates to a nucleic acid molecule containing a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-76. The nucleic acid molecule may also contain any combination of these nucleotide sequences.

In some embodiments, the isolated nucleic acid is DNA.

The nucleic acid molecule of the invention may be isolated from any source that produces a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2. In certain embodiments, the nucleic acid molecule of the invention may be synthesized rather than isolated.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the 07-006 antibody heavy chain variable domain and includes the nucleotide sequence of SEQ ID NO: 77.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of the antibody 07-006 and includes the nucleotide sequence of SEQ ID NO: 78.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the 07-015 antibody heavy chain variable domain and includes the nucleotide sequence of SEQ ID NO: 79.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the 07-015 antibody light chain variable domain and includes the nucleotide sequence of SEQ ID NO: 80.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the 07-016 antibody heavy chain variable domain and includes the nucleotide sequence of SEQ ID NO: 81.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of the antibody 07-016 and includes the nucleotide sequence of SEQ ID NO: 82.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the 07-028 antibody heavy chain variable domain and includes the nucleotide sequence of SEQ ID NO: 83.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of the antibody 07-028 and includes the nucleotide sequence of SEQ ID NO: 84.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain variable domain of the antibody 07-031 and includes the nucleotide sequence of SEQ ID NO: 85.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of the antibody 07-031 and includes the nucleotide sequence of SEQ ID NO: 86.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the 07-041 antibody heavy chain variable domain and includes the nucleotide sequence of SEQ ID NO: 87.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain variable domain of the antibody 07-041 and includes the nucleotide sequence of SEQ ID NO: 88.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the antibody heavy chain 10-001 and includes the nucleotide sequence of SEQ ID NO: 89.

In some embodiments, the nucleic acid is a nucleic acid that encodes the light chain amino acid sequence of the antibody 10-001 and includes the nucleotide sequence of SEQ ID NO: 90.

In some embodiments, the nucleic acid is a nucleic acid that encodes the antibody heavy chain amino acid sequence 10-002 and includes the nucleotide sequence of SEQ ID NO: 91.

In some embodiments, the nucleic acid is a nucleic acid that encodes the light chain amino acid sequence of antibody 10-002 and includes the nucleotide sequence of SEQ ID NO: 92.

In some embodiments, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the antibody heavy chain 10-003 and includes the nucleotide sequence of SEQ ID NO: 93.

In some embodiments, the nucleic acid is a nucleic acid that encodes the light chain amino acid sequence of antibody 10-003 and includes the nucleotide sequence of SEQ ID NO: 94.

In some embodiments, the nucleic acid is a nucleic acid that encodes the antibody heavy chain amino acid sequence 10-007 and includes the nucleotide sequence of SEQ ID NO: 95.

In some embodiments, the nucleic acid is a nucleic acid that encodes the light chain amino acid sequence of the antibody 10-007 and includes the nucleotide sequence of SEQ ID NO: 96.

In some embodiments, the nucleic acid is a nucleic acid that encodes the antibody heavy chain amino acid sequence 10-008 and includes the nucleotide sequence of SEQ ID NO: 97.

In some embodiments, the nucleic acid is a nucleic acid that encodes the light chain amino acid sequence of the antibody 10-008 and includes the nucleotide sequence of SEQ ID NO: 98.

In some embodiments, the nucleic acid is a nucleic acid that encodes the antibody heavy chain amino acid sequence 10-009 and includes the nucleotide sequence of SEQ ID NO: 99.

In some embodiments, the nucleic acid is a nucleic acid that encodes the light chain amino acid sequence of antibody 10-009 and includes the nucleotide sequence of SEQ ID NO: 100.

Nucleic acid molecules can be used to express a recombinant monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2.

Vector

In one aspect, the present invention relates to an expression vector containing the above isolated nucleic acid. The present invention relates to a vector suitable for the expression of any of the nucleotide sequences described herein.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e. a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, the vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, and thus replicate along with the host's genome. Moreover, some vectors are capable of directing the expression of the genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors" ("expression vector" or "expression vector")).

The present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of a monoclonal antibody that specifically binds to GD2, or portions thereof (e.g., heavy chain and/or light chain binding domain sequences) as described herein. The present invention further relates to vectors containing nucleic acid molecules encoding antibodies or fragments of these antibodies.

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YAC, EBV derived episomes, and the like. The DNA molecules can be ligated into the vector such that transcription and translation control sequences in the vector perform their intended function of regulating transcription and translation of the DNA. The expression vector and expression control sequences can be chosen to be compatible with the expression host cell used. DNA molecules encoding part or all of the first and second binding domain sequences (eg, heavy and light chain sequences, where the binding domain contains the heavy and light chain sequence) can be introduced into separate vectors. In one embodiment of the invention, any combination of the above DNA molecules is introduced into the same expression vector. DNA molecules can be introduced into the expression vector by standard methods (eg, ligation of complementary restriction sites on the gene fragment of the antibody and the vector, or blunt-end ligation if no restriction sites are present).

In some embodiments, a suitable vector is one that includes restriction sites such that any VH or VL sequence can be easily incorporated and expressed as described above. Termination of polyadenylation and transcription can occur downstream of the native chromosome site of the encoded regions. The recombinant expression vector may also encode a signal peptide that facilitates production of the antibody chain by the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide of a non-immunoglobulin protein).

In some embodiments, in addition to the antibody gene chain, recombinant expression of the vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as selection of the host cell to be transformed, the level of expression of the desired protein, and so on. Preferred regulatory sequences for a mammalian expression host cell include viral elements that provide high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTR, cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian virus 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

In some embodiments, in addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced.

In some embodiments, the vector may include an expression control sequence. The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie Kozak consensus sequence); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fused cell sequences. host cell

In one aspect, the present invention relates to a method for obtaining a host cell to obtain any of the above antibodies or antigennegative fragment that specifically binds to GD2, and includes transforming the cell with the above vector.

In one aspect, the present invention relates to a host cell for producing any of the above antibodies or antigen-binding fragment that specifically binds to GD2, which contains any of the above nucleic acids.

The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also provides host cells that include, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portions thereof, a nucleotide sequence encoding a light chain or antigen-binding portions thereof, or both of the binding domain of a binding molecule of the present invention. It should be understood that "recombinant host cell" and "host cell" mean not only the specific claimed cell, but also the progeny of such a cell. Because modifications can take place in subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein.

Nucleic acid molecules encoding a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 according to the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or cell, plant or cell, bacterial or yeast cell - owner. The transformation can take place by any known method for introducing the polynucleotides into the host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, nucleic acid-positively charged polymer complex transfection, nucleic acid-calcium phosphate precipitate transfection, polybrene-mediated transfection, protoplast fusion, liposome-encapsulated polynucleotide transfection, and direct microinjection. DNA in the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells with viral vectors.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese Hamster Ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, Hamster Kidney (BHK) cells, African kidney cells green monkeys (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 are introduced into mammalian host cells, the antibodies or fragments thereof are produced by culturing the host cells for a time sufficient to express the antibodies or fragments thereof in the cells. hosts or, more preferably, isolation of antibodies or their fragments in a nutrient medium in which the host cells are grown. A monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2 can be isolated from culture media using standard protein purification techniques. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, and so on. Host bacterial cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Furthermore, the level of production of a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GD2 from a producing cell line can be enhanced by a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions.

It is likely that a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 from different cell lines will differ from each other in the glycosylation profile. However, a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2, encoded by the nucleic acid molecules described herein, or containing the amino acid sequences described herein, are part of this invention, regardless of the glycosylation state of the binding molecules, and in general, regardless on the presence or absence of post-translational modifications.

The above host cell does not refer to a host cell obtained using human embryos.

The above host cell does not refer to a host cell obtained by modifying the genetic integrity of human germline cells.

Method for obtaining an antibody

In one aspect, the present invention relates to a method for producing an antibody or antigen-binding fragment thereof that specifically binds to GD2, which consists in cultivating the above-described host cell in a culture medium under conditions sufficient to obtain the specified antibody or fragment thereof, if necessary, followed by isolating and purifying the resulting antibody or fragment thereof.

The present invention relates to methods for producing a monoclonal antibody or antigen-binding fragment that specifically binds to GD2, according to this invention. One embodiment of the invention relates to a method for producing a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2, as defined herein, comprising obtaining a recombinant host cell capable of expressing a monoclonal antibody or antigen-binding fragment that specifically binds to GD2, culturing said host cell under conditions suitable for expressing the production of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2, and isolating the resulting monoclonal antibody or fragment thereof that specifically binds to GD2. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 obtained by such expression in such recombinant host cells is referred to herein as "recombinant monoclonal antibody that specifically binds to GD2" or "antigen-binding fragment of a recombinant monoclonal antibody that specifically binds to GD2". The invention also relates to the progeny of such host cells.

Pharmaceutical compositions

Another aspect of the invention is a pharmaceutical composition containing as an active ingredient (or as the sole active ingredient) a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GD2.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by GD2, which contains any of the above antibodies or antigen-binding fragments in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

"Pharmaceutical composition" means a composition comprising an antibody according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, auxiliary, distributing and receptive agents, means such as preservatives, stabilizers, fillers, grinders, humectants, emulsifiers, suspending agents, thickeners, sweeteners, flavors, flavors, antibacterial agents, fungicides, lubricants, sustained delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage . Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitan ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as mixtures of these substances. Protection against the action of microorganisms can be provided with a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, and the like. The composition may also include isotonic agents such as sugars, polyols, sodium chloride and the like. Prolonged action of the composition can be provided with agents that slow down the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols and mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of grinders and spreaders are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. Pharmaceutical composition for oral, sublingual, transdermal, intraocular, intramuscular, intravenous, subcutaneous, topical or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard form of administration as a mixture with conventional pharmaceutical carriers . Suitable unit administration forms include oral forms such as tablets, gelatin capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms.

The term "excipient" or "adjuvant" is used herein to describe any component other than an antibody of the invention. These are substances of inorganic or organic origin used in the manufacturing process, the manufacture of medicines to give them the necessary physical and chemical properties.

In some embodiments, the compositions are for the improvement, prevention, or treatment of disorders that may be associated with GD2.

The term "GD2 mediated disease or disorder" refers to all diseases or disorders that are either directly or indirectly related to GD2, including the etiology, development, progression, persistence or pathology of the disease or disorder.

"Treat", "treatment", and "therapy" refer to a method of alleviating or eliminating a biological disorder and/or at least one of its accompanying symptoms. Used herein to "alleviate" a disease, disease, or condition means to reduce the severity and/or frequency of occurrence of symptoms of the disease, disorder, or condition. In addition, references herein to "treatment" include references to curative, palliative, and prophylactic therapy.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female of any age.

The term "disorder" means any condition that can be improved as a result of the treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the occurrence of this disorder.

A “therapeutically effective amount” is the amount of a therapeutic agent administered during treatment that will relieve to some extent one or more of the symptoms of the disease being treated.

In some embodiments of the pharmaceutical composition, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

The pharmaceutical compositions of the present invention and methods for their manufacture will be undeniably obvious to those skilled in the art. The production of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practice) requirements. The composition may include a buffer composition, tonic agents, stabilizers and solubilizers. Prolonged action of the composition can be provided by agents that delay the absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery vehicles are water, ethanol, polyalcohols, and mixtures thereof, oils, and injectable organic esters.

Any method of administering peptides, proteins, or antibodies accepted in the art can be appropriately used for a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2 of the present invention.

The term "pharmaceutically acceptable" means one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.

The term “buffer”, “buffer composition”, “buffer agent” refers to a solution capable of maintaining the pH value due to the interaction of the acidic and alkaline components that make up its composition, which enables the antibody preparation that specifically binds to GD2 to show resistance to pH changes. In general, pH values of the pharmaceutical composition between 4.0 and 8.0 are preferred. As buffer agents, for example, acetate, phosphate, citrate, histidine, succinate, and the like can be used. buffer solutions, but not limited to.

The terms "tonic agent", "osmolytic" or "osmotic agent", as used here, refer to an excipient that can supply the osmotic pressure of a liquid antibody preparation. An "isotonic" drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic formulations typically have an osmotic pressure of about 250 to 350 mOsm/kg. As isotonic agents, polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, and the like can be used, but not limited to.

By "stabilizer" is meant an excipient or a mixture of two or more excipients which provide physical and/or chemical stability to the active agent. As stabilizers can be used amino acids, for example, arginine, histidine, glycine, lysine, glutamine, proline, but not limited to; surfactants, e.g. polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxaner, Pluronic), sodium dodecyl sulfate (SDS) ), but not limited to; antioxidants, for example, methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of sulfuric acids, and the like, but not limited to; chelating agents such as, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, and the like.

The pharmaceutical composition according to the invention is stable.

A pharmaceutical composition is "stable" if the active agent retains its physical stability and/or chemical stability and/or biological activity during the stated shelf life at a storage temperature, for example, at 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The storage period is selected based on the results of a stability study with accelerated and natural storage.

The pharmaceutical composition of the present invention may be formulated, packaged, or widely sold as a single unit dose or multiple unit dose units in the form of a finished dosage form. As used herein, the term "unit dosage unit" means a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is usually equal to the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such dosage, for example, half or one third of such dosage.

Pharmaceutical compositions of the present invention are generally suitable for parenteral administration as sterile medicaments intended to be administered to the human body in violation of the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by injection, infusion or implantation. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intraarticular, transdermal injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be useful. Regional perfusion is also provided. Preferred embodiments of the invention include intravenous and subcutaneous routes. Any method of administering peptides or proteins accepted in the art can be appropriately used for an antibody or antigen-binding fragment thereof that specifically binds to GD2 of the present invention.

Injectable drugs can be manufactured, packaged or sold in unit dosage form, such as, but not limited to, ampoules, vials, plastic containers, pre-filled syringes, auto-injection devices. Drugs for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

Another embodiment of the invention is a drug for parenteral administration, where the pharmaceutical composition is provided in dry form, i.e., powder or granules for dissolution in a suitable solvent (eg, sterile, pyrogen-free water) prior to administration. Such a drug can be obtained, for example, by lyophilization, i. a process known in the art as freeze-drying, which involves freezing the preparation and then removing the solvent from the frozen contents.

An antibody or antigen-binding fragment thereof that specifically binds to GD2 of the present invention may also be administered intranasally or by inhalation, alone, as a mixture with a suitable pharmaceutically acceptable vehicle from an inhaler, such as a pressurized aerosol container, pump, spray, nebulizer or nebulizer. , which uses or does not use a suitable propellant, or in the form of nasal drops or spray.

The drug for parenteral administration may be immediate or modified release. Modified release drugs include delayed, sustained, pulsatile, controlled, targeted and programmed release.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by GD2, which contains any of the above antibody or antigennegative fragment and at least one other therapeutically active compound.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an antibody, a chemotherapeutic agent, or a hormone therapy agent.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an immune checkpoint inhibitor.

In some embodiments of the pharmaceutical composition, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In some embodiments of the pharmaceutical composition, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments of the pharmaceutical composition, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the pharmaceutical composition, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments of the pharmaceutical composition, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab.

In some embodiments of the pharmaceutical composition, the PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

In some embodiments of the pharmaceutical composition, the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

Therapeutic use of a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2

In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to GD2 is used in the treatment of disorders mediated by GD2 activity.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The aforementioned subject may be male or female and of any age.

In the case of a tumor (eg, a cancerous tumor), a therapeutically effective amount of an antibody or antibody fragment (eg, an antibody or antibody fragment that specifically binds GD2) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (ie, slow down to some extent and preferably stop) cancer cell infiltration of peripheral organs; inhibit (ie, slow down to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or alleviate, to some extent, one or more of the symptoms associated with the disorder. The antibody or antibody fragment may, to some extent, prevent the growth and/or kill existing cancer cells, it may cause a cytostatic and/or cytotoxic effect. In cancer therapy, in vivo efficacy can be determined, for example, by evaluating survival, time to disease progression (TTR), tumor response rate (RR), duration of response, and/or quality of life.

As used herein, uses or methods relating to an antibody, or antigen-binding fragment thereof, that specifically binds to GD2, to one or more other therapeutic agents, are intended to mean, refer to, or include:

1) simultaneous administration of such a combination of an antibody or antigen-binding fragment thereof that specifically binds to GD2 and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which these components are released almost simultaneously to the specified patient,

2) simultaneous administration of such a combination of an antibody or antigen-binding fragment thereof that specifically binds to GD2 and a therapeutic agent to a patient in need of treatment, when such components are formulated separately in different dosage forms, the introduction of which occurs at almost the same time to the specified patient , after which these components are released almost simultaneously to the specified patient,

3) sequential administration of such a combination of an antibody or antigen-binding fragment thereof that specifically binds to GD2 and a therapeutic agent to a patient in need of treatment, when such components are formulated separately from each other in separate dosage forms that are taken in sequential time by said patient with a significant time interval between each administration, after which these components are released at practically different times to the specified patient; and

4) sequential administration of such a combination of an antibody or antigen-binding fragment thereof that specifically binds to GD2 and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which the release of these components occurs in a controlled manner, after which they simultaneously, sequentially or jointly released at the same time and/or different times to the specified patient, where each part can be entered in one or different ways.

An antibody or antigen-binding fragment thereof that specifically binds to GD2 can be administered without additional therapeutic treatment, i. as a standalone therapy.

In one aspect, the present invention provides a method for inhibiting GD2 biological activity in a subject in need of such inhibition, which comprises administering to the subject an effective amount of any of the foregoing antibodies or antigen-binding fragment thereof.

In one aspect, the present invention relates to a method of treating a disease or disorder mediated by GD2, comprising administering to a subject in need of such treatment, any of the above antibodies or antigen-binding fragment or the above pharmaceutical composition in a therapeutically effective amount.

In one aspect, the present invention relates to a method of treating a disease or disorder mediated by GD2, which includes administering to a subject in need of such treatment, any of the above antibodies or antigen-binding fragments and selected from the group:

a) administering at least one other therapeutically active compound,

b) radiation therapy,

c) transplantation of hematopoietic stem cells,

d) surgical treatment and, if necessary, adjuvant therapy,

or

e) any combination of the above a)-d).

In some embodiments of the method of treatment, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma , Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

In some embodiments of the method of treatment, the other therapeutically active compound is an antibody, a chemotherapeutic agent, or a hormone therapy agent.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (eg bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARTNOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-amino camptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediine antibiotics (e.g., calicheamicin, e.g., calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin, including dynemycin A; esperamycin; as well as neocarcinostatin chromophore and related chromoprotein chromophores - enediine antibiotics, aclacinomysins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorub icin, 6-diazo-5-oxo -L-norleucine, doxorubicin (including ADRIAMICIN®, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicinoNSL in injectable liposomes (DOXOL®), TLC D-99 liposomal doxorubicin (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; adrenal suppressants such as aminoglutethimide, mitotane, trilostane; folic acid compensator such as folinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diazichon; elfornitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubipine; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (eg, T-2 toxin, verracurin A, roridin A, and anguidin); urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside ("ara-C"); thiotepa; a taxoid, eg paclitaxel (TAXOL®), an engineered albumin-bound nanoparticle formulation of paclitaxel (ABRAXANETM) and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin and carboplatin; vinca alkaloids that prevent the polymerization of tubulin from resulting microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantron; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (e.g. BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID ®) or risedronate (ACTONEL®); troxcitabine (1,3-dioxolane nucleoside analog of cytosine); antisense oligonucleotides, eg oligonucleotides that inhibit gene expression in signaling pathways involved in aberrant cell proliferation such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; a topoisomerase 1 inhibitor (eg LURTOTECAN®); rmRH (eg ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosin, COX-2 inhibitor (eg celecoxib or etoricoxib), proteasome inhibitor (eg PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor such as sodium oblimersene (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above, such as CHOP, short for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, short for oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovorin.

Hormonal agents are agents that act to regulate or inhibit the action of hormones on tumors. Examples of such agents include antiestrogen with a mixed agonist/antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxy tamoxifen, trioxifene, toremifene (FARESTON®); idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure antiestrogen without agonist properties such as fulvestrant (FASLODEX®) and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER metabolism and/or decrease ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASFN®), and non-steroidal aromatase inhibitors such as anastrozole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide and other aromatase inhibitors, including vorozol (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazole; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all-trans retinoic acid and fenretinide; onapriston; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above means.

In some embodiments of the method of treatment, the other therapeutically active compound is an immune checkpoint inhibitor.

The term "immune checkpoint inhibitor" (or checkpoint inhibitor) refers to compounds that inhibit the activity of immune checkpoints. Inhibition includes reduced function or complete blockade. Examples of inhibitory checkpoint molecules include B7-H3, B7-H4, BTLA, CTLA-4, KIR, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, TIGIT and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. A number of immune checkpoint inhibitors are known and alternative immune checkpoint inhibitors similar to these known inhibitors of immune checkpoint proteins may be developed in the near future. Immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molecules, and small molecules.

In some embodiments of the method of treatment, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In some embodiments of the method of treatment, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1. Examples of antibodies that specifically bind to PD-1 include pembrolizumab (pembrolizumab), nivolumab (mvolumab), prolgolimab (prolgolimab), toripalimab (toripalimab), cemiplimab (cemiplimab), sintilimab (sintilimab) and others. Most preferred is prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the method of treatment, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments of the method of treatment, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. Examples of antibodies that specifically bind to CTLA4 include ipilimumab (ipilimumab), tremelimumab (tremelimumab), zalifrelimab (zalifrelimab), nurulimab, and others. Most preferred is ipilimumab or nurulimab.

In some embodiments of the method of treatment, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab.

In some embodiments of the method of treatment, the PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

In some embodiments of the treatment method, the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments of the method of treatment, the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

In one aspect, the present invention relates to the use of the above antibody or antigen-binding fragment or the above pharmaceutical composition for the treatment of a GD2 mediated disease or disorder in a subject in need of such treatment.

In one aspect, the present invention relates to the use of any of the above antibodies or antigen-binding fragment and at least one of the group:

a) another therapeutically active compound,

b) radiation therapy,

c) transplantation of hematopoietic stem cells or

d) surgical treatment and, if necessary, adjuvant therapy, for the treatment of a disease or disorder mediated by GD2.

In some embodiments, the GD2 mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma.

In some embodiments, the other therapeutically active compound is an immune checkpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments, the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab.

In some embodiments, the PD-L1 inhibitor is an antibody that specifically binds to PD-L1.

In some embodiments, the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab.

In some embodiments, the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

Doses and routes of administration

The monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to GD2 of the present invention will be administered in an amount effective to treat the condition in question, i. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether administration of a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GD2 is self-treatment or carried out in combination with one or more additional drugs or treatments.

Drug regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the severity of the therapeutic situation. Especially useful is the formulation of parenteral compositions in unit dosage form for ease of administration and uniformity of dosing. Unit dosage form, as used herein, refers to physically discrete units suitable as unit doses for patients/subjects to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification for unit dosage forms of the present invention is generally dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for sensitivity treatment in subjects.

Thus, those skilled in the art will appreciate from the disclosure provided herein that doses and dosing regimens are adjusted in accordance with methods well known in the therapeutic field. This means that the maximum tolerated dose can be easily set and the effective amount that provides a detectable therapeutic effect for the patient can also be determined, as well as the time requirements for the administration of each agent to achieve a visible therapeutic effect for the patient. Thus, although some doses and regimens of administration are given as examples in this document, these examples in no way limit the doses and regimens of administration that may be needed for a patient in the practice of the present invention.

It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated and may include one or more doses. In addition, it should be understood that for any particular patient, specific administration schedules will need to be adjusted over time according to individual need and at the discretion of the healthcare professional who administers or supervises the administration of the compositions, and that the concentration ranges given in this specification are only by way of example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen with the compositions of this invention may be based on various factors, including the type of disease, age, weight, sex, health status of the patient, severity of the condition, route of administration, and the specific monoclonal antibody or antigen-binding fragment used that specifically binds to GD2. Thus, the dosing regimen may vary widely, but may be determined regularly using standard methods. For example, doses may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the present invention encompasses individual dose escalation as determined by the skilled artisan. Determination of the required dose and modes are well known in the relevant field of technology and will be clear to a person skilled in the art after reading the ideas disclosed in this document.

Examples of suitable routes of administration are provided above.

It is contemplated that a suitable dose of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 of the present invention will be in the range of 0.1-200 mg/kg, preferably 0.1-100 mg/kg, including about 0. 5-50 mg/kg, for example about 1-20 mg/kg. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 may be administered, for example, at a dose of at least 0.25 mg/kg, for example, at least 0.5 mg/kg, including at least 1 mg /kg, for example, at least 1.5 mg/kg, for example, as well as at least 2 mg/kg, for example, at least 3 mg/kg, including at least 4 mg/kg, for example, according to at least 5 mg/kg; and for example up to a maximum of 50 mg/kg, including up to a maximum of 30 mg/kg, for example up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. The administration will be repeated usually at suitable intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as is deemed appropriate by the physician in charge, who may in some cases increase or reduce dose if necessary.

Diagnostic use of an antibody that specifically binds to GD2

The monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 of the present invention is also useful for diagnostic purposes (eg, in vitro, ex vivo). For example, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 of the invention can be used to detect or measure the level of GD2 in samples obtained from a patient, such as a tissue sample or a body fluid sample such as inflammatory exudate, blood, blood serum, intestinal fluid, saliva or urine. Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay, radioimmunoassay, and immunohistology.

Examples

For a better understanding of the invention, the following examples are given. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents, and patent applications referenced in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example in order to avoid ambiguous interpretation, it will be quite clear to those skilled in the art based on the ideas disclosed in this invention that certain changes and modifications can be made without deviating from the essence and scope of the attached options. implementation of the invention. Materials and General Methods

General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is provided by: Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Antibody chain amino acids are numbered according to EU numbering (Edelman G.M. et al., Proc Natl Acad Sci USA 63 1969 ee 78-85 Kabat E A et al Sequences of Proteins of Immunological Interest 5th ed Public Health Service National Institutes of Health, Bethesda, MD, 1991).

Recombinant DNA methods

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

Gene synthesis

The desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging in length from 300 to 1400 bp, which are flanked by unique restriction sites, were assembled by annealing and ligation of oligonucleotides, including PCR amplification and subsequent cloning through restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

Determination of DNA sequences

DNA sequences were determined by Sanger sequencing.

Analysis of DNA and protein sequences and processing of sequence data

The Unipro UGENE software package version 1.29 and SnapGene Viewer were used to create, map, analyze, annotate, and illustrate sequences.

Expression vectors

For the expression of the antibodies described in the application materials, variants of expression plasmids intended for the expression of antibodies in prokaryotic cells (E. coli) of short-term expression in eukaryotic cells (for example, in CHO cells) were used. In addition to the expression cassette of the antibody, the vectors contained: a replication initiation site that ensures the replication of the indicated plasmid in E. coli, genes that confer resistance in E. coli to various antibiotics (for example, to ampicillin, kanamycin).

Fusion of genes containing the described antibody chains, as indicated below, was carried out by PCR and/or gene synthesis and assembly using known methods and recombination procedures by joining appropriate nucleic acid segments, for example, using unique restriction sites in appropriate vectors. Subcloned nucleotide sequences were confirmed by DNA sequencing. For short-term transfections, large numbers of plasmids were generated by obtaining plasmids from transformed E. coli cultures.

Example 1

Selection of annr-GD2 antibody sequences

The structural data obtained in silico were the material for the creation of anra-GD2 antibody molecules. Scaffolding in silico was carried out using the internal algorithm of ZAO Biocad. Structures were prepared using the Prep Wizard tool from Schrodinger Suite 2017-2. After that, folding was carried out using the Prime tool from Schrodinger Suite 2017-2.

An approach was applied to sequentially change the amino acid composition of variable domains.

Antibodies were optimized in silico, thus, antibody candidates for further studies were obtained, which are indicated in table 1.

From table 1 were selected 6 leader antibodies: 07-006, 07-015, 07-016, 07-028, 07-031, 07-041, which unexpectedly had the best parameters (see examples below).

Example 2

Analysis of the identity and humanization of variable fragments of the heavy and light chains of anra-GD2 antibodies 07-006, 07-015, 07-016, 07-028, 07-031 or 07-041

Table 2 shows the identity analysis of the heavy chain variable fragments of anra-GD2 antibodies 07-006, 07-015,07-016,07-028, 07-031, or 07-041.

Thus, the anra-GD2 heavy chain variable fragments of the invention share at least 98 percent identity with each other.

Table 3 shows the humanization analysis of the heavy chain variable fragments of anra-GD2 antibodies 07-006, 07-015, 07-016, 07-028, 07-031, or 07-041.

Thus, the heavy chain variable fragments of the anra-GD2 antibodies of the invention are more than 80 percent humanized.

Table 4 shows the identity analysis of the anra-GD2 light chain variable fragments of antibodies 07-006, 07-015, 07-016, 07-028, 07-031 or 07-041

Thus, the variable light chain fragments of the anra-GD2 antibodies of the invention have at least 96 percent identity with each other.

Table 5 shows the humanization analysis of the anra-GD2 light chain variable fragments of antibodies 07-006, 07-015, 07-016, 07-028, 07-031, or 07-041.

Thus, the anti-GD2 candidate light chain variable fragments of the invention are more than 80 percent humanized.

Example 3

Obtaining anra-GD2 sequences of antibodies 07-006, 07-015, 07-016, 07-028, 07-031 or 07-041

The genes for the heavy and light chain variable domains of an anti-GD2 antibody of the invention, which is selected from the group: 07-006, 07-015,07-016,07-028,07-031, or 07-041, were synthesized de novo. For this, oligonucleotides of 55-60 n.o. were synthesized. each forming a completely overlapping gene sequence. Each gene was assembled by two-round PCR, resulting in 339 bp fragments. every. The fusion of the heavy chain variable domain gene and the human IgGl Fc fragment, the light chain variable domain and SC was performed by PCR and/or gene synthesis and assembly using known recombination methods and procedures by joining the corresponding nucleic acid segments, for example, using SOE- PCR (Splicing by overlap extension).

The heavy and light chain genes of an anti-GD2 antibody of the invention, which is selected from the group: 07-006, 07-015, 07-016, 07-028, 07-031, or 07-041, were cloned into pEE plasmids to generate protein in the format IgGl in mammalian cells. The cloned nucleotide sequences were confirmed by DNA sequencing. The resulting plasmids (Figures 2 and 3) were obtained in the required quantities in E. coli cells and purified using a commercial Qiagen plasmid DNA isolation kit.

The resulting genetic constructs were transferred for transient production of proteins in the CHO cell line.

Example 4

Modification of the Fc heavy chain constant domain of antibodies aGD2 07-006, 07-015, 07-016, 07-028, 07-031 or 07-041

To obtain antibodies with improved properties, the Fc heavy chain constant domain was modified by introducing point mutations M252Y, S254T, T256 (YTE) and/or K322A. A set of YTE mutations makes it possible to achieve prolonged pharmacokinetics, and the introduction of the K3 22 A mutation reduces the complement-dependent cytotoxicity of the obtained antibodies. Also, due to the expression in the cell line CHO-lg6-Fut8, afucosylation of the Fc part of the antibody occurred, which gives an increase in antibody-dependent cellular cytotoxicity. The antibodies obtained are shown in Table 6.

The assembly of genetic constructs included the fusion of the heavy chain variable domain gene and human IgGl Fc, which was previously introduced with point mutations.

The heavy chain genes with substitutions were cloned into pEE plasmids for protein production, together with already obtained light chain constructs, in the IgGl format in the CHO-lg6-Fut8 cell line. The cloned nucleotide sequences were confirmed by DNA sequencing. The resulting plasmids (Figure 2, 3) were produced in the required quantities in E. coli cells and purified using the Qiagen kit.

The resulting genetic constructs were transferred for transient production of proteins in the CHO-lg6-Fut8 cell line.

Example 5

Obtaining, isolation and purification of antibodies to GD2 from a suspension culture of mammalian cells.

Full-length antibodies were produced in CHO cell growth medium, afucosylated full-length antibodies were produced in CHO-lg6-Fut8 cell growth medium. After transfection of cells with expression vectors, orbital feed-batch cultivation was performed in a serum-free medium for 7 days. The secretion of the studied antibodies was controlled using the Pall ForteBio Octet RED 96 molecular interaction analysis system on proteinA biosensors.

At the end of cultivation, the medium was centrifuged at 2000 g for 20 minutes and filtered through a filter with a pore size of 0.22 μm. Target proteins were isolated from the culture liquid by affinity chromatography on an Akta Pure 25 chromatographic system using HiTrap rProtein A FF columns. The culture fluid was applied to a HiTrap rProtein A FF column, after which the column was washed with PBS and the protein was eluted with a solution of 0.1 M glycine buffer pH 3, after which the protein solution was neutralized by adding 1 M Tris-HCl pH8 in a ratio of 1/5 v\V. Next, the protein was transferred into PBS pH 7.4 using dialysis, after which the resulting solution was filtered (0.22 μm). The preparation was stored at -70°C. The purity of the resulting protein solution was assessed using SDS-gel electrophoresis (figure 4).

Example 6

Kinetic studies of the affinity of anti-GD2 antibodies with GD2 ganglioside using Forte Bio OctetRed96.

The binding of the antibody to GD2 was assessed by biolayer interferometry on an OctetRed96 instrument (Pall). AR2G sensors were coated with GD2 ganglioside. Then the sensors with immobilized GD2 were immersed in the wells with the antibody. After association of the antibody and ganglioside, the sensors were immersed in the working solution for the subsequent dissociation step. The obtained sensograms with the subtraction of the base signal were analyzed using the Octet Data Analysis program (version 8.2) according to the standard procedure using the 1:1 interaction model. KD for aGD2 antibodies of the invention are shown in Table 7.

Thus, all analyzed aHTH-GD2 antibodies specifically bind to ganglioside GD2 (table 7) with high affinity. Example 7

Analysis of the thermal stability of antibodies to GD2.

Antibodies were heated in PBS pH 7.4 using an amplifier in plastic test tubes at 50°C for 48 hours, followed by a transition to +4°C. After the end of the program, samples were analyzed before and after heating using analytical gel chromatography on a TSK Gel G3000 SWxl column. Compared the area of the target peaks of the samples before and after heating. A change of less than 5% of the monomer peak area after heating for 48 hours indicates the stability of the drug and the possibility of long-term storage (table 8).

Thus, all analyzed aHTH-GD2 antibodies have high thermal stability (table 8). Example 8

Determination of antibody-dependent cellular cytotoxicity of antibodies to

GD2

A reporter cell line based on the Jurkat cell line, stably expressing CD 16 on the surface and containing the gene encoding firefly luciferase under the control of the NFAT promoter, was used; SK-N-BE(2) was used as target cells. Jurkat-NFAT-Luc-CD16 cells were cultured at 37°C 5% CO ₂ in RPMI-1640 medium (10% FBS, 10 μg/ml gentamicin, 2 mM L-glutamine, 0.3 μg/ml puromycin and 200 μg/ml ml of hygromycin), SK-N-BE(2) was cultured under the same conditions in DMEM/F12 medium (10% FBS, 10 µg/ml gentamicin, and 2 mM L-glutamine).

In each well of a 96-well culture plate, 25,000 Jurkat-NFAT-Luc-CD16 effector cells and 25,000 SK-N-BE(2) target cells were added in a volume of 50 μl, as well as antibody dilutions at the concentration indicated on the graph in volume of 50 µl.

Points without antibody addition were used as a negative control. The plates were incubated for 4 hours at 37°C, 5% CO ₂ and further, using a substrate for luciferase (ZAO BIOCAD), the luminescence intensity in the wells was measured on a Spark plate reader (Tecan), data processing and plotting were performed using software SigmaPlot 14.0 software. EC ₅₀ parameter values for anti-GD2 antibody samples are shown in Table 9.

Figure 5 shows that antibody 10-008 has antibody-dependent cellular cytotoxicity in the test on the Jurkat-NFAT-Luc-CD 16 reporter cell line.

Example 9

Complement dependent cellular cytotoxicity assay

The SK-N-BE (2) (human neuroblastoma) line was used as target cells for analysis. Cells were grown in DMEM medium with 10% bovine serum. For the analysis, a cell suspension was prepared in DMEM medium with 0.1% bovine serum albumin at a concentration of 1x10 ⁶ cells/ml, and a number of dilutions of the studied antibody candidates were also prepared.

50 μl of antibody dilutions, 50 μl of cell suspension, and 50 μl of freshly isolated human serum taken from healthy donors were added to a 96-well culture plate. The tablet was incubated at 37°C, 5% CO ₂ for 3 hours. After completion of incubation, 15 µl of Alamar Blue vital dye (Invitrogen) was added to all wells and the plate was left to incubate at 37°C, 5% CO2 for 18 h.

Shake the tablet for 10-20 minutes at room temperature on an orbital shaker to mix. Fluorescence readings were measured using a TECAN Spark plate reader. The detected fluorescence signal is proportional to the number of viable cells. For data processing and plotting, Excel and SigmaPlot 14.0 software was used.

Investigated antibodies 07-041 dFuc ((afucosylated version 07-041)), 10-007, 10-008, 10-009 cause the death of target cells in the presence of human serum. Antibodies 10-008 and 10-009 have a reduced effect compared to other antibodies (see figure 6).

Claims (136)

1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to GD2 (GD2 ganglioside), comprising: (a) a heavy chain variable domain comprising: (i) a CDR1 with an amino acid sequence selected from the group: SEQ ID NO: 1 or SEQ ID NO: 2, (ii) CDR2 with the amino acid sequence of SEQ ID NO: 3, (iii) a CDR3 with an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; And (b) a light chain variable domain comprising: (i) a CDR1 with an amino acid sequence selected from the group: SEQ ID NO: 8 or SEQ ID NO: 9, (ii) a CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 10 or SEQ ID NO: 11, (iii) a CDR3 with an amino acid sequence selected from the group: SEQ ID NO: 12 or SEQ ID NO: 13. 2. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains: (i) FR1 with the amino acid sequence of SEQ ID NO: 42, (ii) FR2 with the amino acid sequence of SEQ ID NO: 43, (iii) FR3 with the amino acid sequence of SEQ ID NO: 44 and (iv) FR4 with the amino acid sequence of SEQ ID NO: 45. 3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain contains: (i) FR1 with the amino acid sequence of SEQ ID NO: 46, (ii) FR2 with an amino acid sequence selected from the group: SEQ ID NO: 47 or SEQ ID NO: 48, (iii) FR3 with the amino acid sequence of SEQ ID NO: 49 and (iv) FR4 with the amino acid sequence of SEQ ID NO: 50. 4. The isolated monoclonal antibody or its antigen-binding fragment according to claim 1, where a) the heavy chain variable domain contains: (i) FR1 with the amino acid sequence of SEQ ID NO: 42, (ii) FR2 with the amino acid sequence of SEQ ID NO: 43, (iii) FR3 with the amino acid sequence of SEQ ID NO: 44 and (iv) FR4 with the amino acid sequence of SEQ ID NO: 45, and where b) the light chain variable domain contains: (i) FR1 with the amino acid sequence of SEQ ID NO: 46, (ii) FR2 with an amino acid sequence selected from the group: SEQ ID NO: 47 or SEQ ID NO: 48, (iii) FR3 with the amino acid sequence of SEQ ID NO: 49 and (iv) FR4 with the amino acid sequence of SEQ ID NO: 50. 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains: (i) CDR1 with the amino acid sequence of SEQ ID NO: 2, (ii) CDR2 with the amino acid sequence of SEQ ID NO: 3, (iii) CDR3 with the amino acid sequence of SEQ ID NO: 5. 6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains: (i) CDR1 with the amino acid sequence of SEQ ID NO: 2, (ii) CDR2 with the amino acid sequence of SEQ ID NO: 3, (iii) CDR3 with the amino acid sequence of SEQ ID NO: 4. 7. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain contains: (i) CDR1 with the amino acid sequence of SEQ ID NO: 9, (ii) CDR2 with the amino acid sequence of SEQ ID NO: 11, (iii) CDR3 with the amino acid sequence of SEQ ID NO: 12. 8. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain contains: (i) CDR1 with the amino acid sequence of SEQ ID NO: 8, (ii) CDR2 with the amino acid sequence of SEQ ID NO: 10, (iii) CDR3 with the amino acid sequence of SEQ ID NO: 12. 9. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains an amino acid sequence that has at least 98 percent identity with the amino acid sequence of SEQ ID NO: 17. 10. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain contains an amino acid sequence selected from the group: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. 11. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain contains an amino acid sequence that has at least 96 percent identity with the amino acid sequence of SEQ ID NO: 21. 12. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain contains an amino acid sequence selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. 13. An isolated monoclonal antibody or its antigen-binding fragment according to claim 1, where: (a) the heavy chain variable domain contains an amino acid sequence that has at least 98 percent identity with the amino acid sequence of SEQ ID NO: 17; (b) the light chain variable domain contains an amino acid sequence that has at least 96 percent identity with the amino acid sequence of SEQ ID NO: 21. 14. An isolated monoclonal antibody or its antigen-binding fragment according to claim 1, where: (a) the heavy chain variable domain contains an amino acid sequence selected from the group: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; (b) the light chain variable domain contains an amino acid sequence selected from the group: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. 15. An isolated monoclonal antibody or its antigen-binding fragment according to claim 14, where: (a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 16; (b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 19. 16. An isolated monoclonal antibody or its antigen-binding fragment according to claim 14, where: (a) the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 17; (b) the light chain variable domain contains the amino acid sequence of SEQ ID NO: 21. 17. Selected monoclonal antibody according to any one of paragraphs. 1-16, wherein the antibody that specifically binds to GD2 is a full length IgG antibody. 18. The isolated monoclonal antibody of claim 17, wherein the full-length IgG antibody is of the human IgG1, IgG2, IgG3, or IgG4 isotype. 19. The isolated monoclonal antibody of claim 18, wherein the full length IgG antibody is of the human IgG1 isotype. 20. An isolated monoclonal antibody according to claim 1, wherein the antibody contains YTE (M252Y, S254T, T256E) and/or K322A mutations in the Fc fragment compared to the natural sequence of the Fc fragment. 21. An isolated monoclonal antibody according to claim 1, comprising a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO : 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 , SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. 22. An isolated monoclonal antibody according to claim 1, comprising a light chain containing an amino acid sequence selected from the group: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. 23. An isolated monoclonal antibody according to claim 1, including: (a) a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, and (b) a light chain containing an amino acid sequence selected from the group: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. 24. An isolated monoclonal antibody according to claim 23, including: (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 32, and (b) a light chain containing the amino acid sequence of SEQ ID NO: 39. 25. An isolated monoclonal antibody according to claim 23, including: (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 33, and (b) a light chain containing the amino acid sequence of SEQ ID NO: 41. 26. Selected nucleic acid that encodes an antibody or antigennegative fragment according to any one of paragraphs. 1-25. 27. The isolated nucleic acid of claim 26, wherein the nucleic acid is DNA. 28. An expression vector containing a nucleic acid according to any one of paragraphs. 26, 27. 29. The method of obtaining a host cell to obtain an antibody or antigennegative fragment according to any one of paragraphs. 1-25, which includes transforming the cell with the vector of claim 28. 30. The host cell to obtain an antibody or antigennegative fragment according to any one of paragraphs. 1-25 containing a nucleic acid according to any one of paragraphs. 26, 27. 31. The method of obtaining an antibody or antigennegative fragment according to any one of paragraphs. 1-25, which consists in culturing the host cell according to claim 30 in a culture medium under conditions sufficient to obtain the specified antibody, if necessary, followed by isolation and purification of the resulting antibody. 32. Pharmaceutical composition for the treatment of a disease or disorder mediated by GD2, containing an antibody or antigennegative fragment according to any one of paragraphs. 1-25 in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients. 33. The pharmaceutical composition according to claim 32, wherein the disease or disorder mediated by GD2 is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma. 34. Pharmaceutical composition for the treatment of a disease or disorder mediated by GD2, containing an antibody or antigennegative fragment according to any one of paragraphs. 1-25 and at least one other therapeutically active compound. 35. The pharmaceutical composition according to claim 34, wherein the disease or disorder mediated by GD2 is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma. 36. Pharmaceutical composition according to any one of paragraphs. 34, 35, wherein the other therapeutically active compound is an antibody, chemotherapeutic agent, or hormone therapy agent. 37. Pharmaceutical composition according to any one of paragraphs. 34, 35, wherein the other therapeutically active compound is an immune checkpoint inhibitor. 38. The pharmaceutical composition of claim 37, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. 39. The pharmaceutical composition of claim 38, wherein the PD-1 inhibitor is an antibody that specifically binds to PD-1. 40. The pharmaceutical composition according to claim 39, wherein the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab. 41. The pharmaceutical composition of claim 38, wherein the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. 42. The pharmaceutical composition of claim 41, wherein the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab. 43. The pharmaceutical composition of claim 38, wherein the PD-L1 inhibitor is an antibody that specifically binds to PD-L1. 44. The pharmaceutical composition according to claim 43, wherein the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab. 45. Pharmaceutical composition according to any one of paragraphs. 34, 35, where the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group. 46. A method of inhibiting the biological activity of GD2 in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-25. 47. A method of treating a disease or disorder mediated by GD2, comprising administering to a subject in need of such treatment, an antibody or antigen-binding fragment according to any one of paragraphs. 1-25 or a pharmaceutical composition according to any one of paragraphs. 32-45 in a therapeutically effective amount. 48. A method of treating a disease or disorder according to claim 47, wherein the GD2-mediated disease or disorder is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, carcinoma kidney, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma. 49. A method of treating a disease or disorder mediated by GD2, comprising administering to a subject in need of such treatment, an antibody or antigen-binding fragment according to any one of paragraphs. 1-25 and selected from the group: a) administering at least one other therapeutically active compound, b) radiation therapy, c) transplantation of hematopoietic stem cells, d) surgical treatment and, if necessary, adjuvant therapy or e) any combination of the above a)-d). 50. A method of treating a disease or disorder according to claim 49, wherein the disease or disorder mediated by GD2 is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, carcinoma kidney, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma. 51. A method of treating a disease or disorder according to any one of paragraphs. 49, 50, wherein the other therapeutically active compound is an antibody, chemotherapeutic agent, or hormone therapy agent. 52. A method of treating a disease or disorder according to any one of paragraphs. 49, 50, wherein the other therapeutically active compound is an immune checkpoint inhibitor. 53. A method of treating a disease or disorder according to claim 52, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. 54. A method of treating a disease or disorder according to claim 53, wherein the PD-1 inhibitor is an antibody that specifically binds to PD-1. 55. A method of treating a disease or disorder according to claim 54, wherein the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab. 56. A method of treating a disease or disorder according to claim 53, wherein the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. 57. A method of treating a disease or disorder according to claim 56, wherein the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab. 58. A method of treating a disease or disorder according to claim 53, wherein the PD-L1 inhibitor is an antibody that specifically binds to PD-L1. 59. A method of treating a disease or disorder according to claim 58, wherein the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab. 60. A method of treating a disease or disorder according to any one of paragraphs. 49-50, wherein the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group. 61. The use of an antibody or antigen-binding fragment according to any one of paragraphs. 1-25 or a pharmaceutical composition according to any one of paragraphs. 32-45 for treating a GD2 mediated disease or disorder in a subject in need of such treatment. 62. Use according to claim 61, wherein the disease or disorder mediated by GD2 is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma. 63. The use of an antibody or antigen-binding fragment according to any one of paragraphs. 1-25 and at least one of the group: a) another therapeutically active compound, b) radiation therapy, c) transplantation of hematopoietic stem cells or d) surgical treatment and, if necessary, adjuvant therapy, for the treatment of a disease or disorder mediated by GD2. 64. Use according to claim 63, wherein the disease or disorder mediated by GD2 is selected from the group: brain tumor, neuroblastoma, glioblastoma, medulloblastoma, retinoblastoma, astrocytoma, melanoma, B-cell lymphoma, small cell lung cancer, renal carcinoma, desmoplastic small round cell fibroma, osteosarcoma, Ewing's sarcoma, breast cancer, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, or soft tissue sarcoma. 65. Application according to any one of paragraphs. 63, 64, wherein the other therapeutically active compound is an immune checkpoint inhibitor. 66. The use of claim 65, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. 67. The use of claim 66 wherein the PD-1 inhibitor is an antibody that specifically binds to PD-1. 68. Use according to claim 67, wherein the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab. 69. The use of claim 66 wherein the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. 70. Use according to claim 69, wherein the antibody that specifically binds to CTLA-4 is ipilimumab or nurulimab. 71. The use of claim 66 wherein the PD-L1 inhibitor is an antibody that specifically binds to PD-L1. 72. Use according to claim 71, wherein the antibody that specifically binds to PD-L1 is selected from the group: durvalumab, avelumab, atezolizumab, manelimab. 73. Application according to any one of paragraphs. 63, 64, where the other therapeutically active compound is selected from the group: IL-2, GM-CSF, isotretinoin, one or more other cytokines, or any combination of therapeutically active compounds from this group.

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