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CN118620082A - Bispecific antigen binding proteins targeting the VEGF pathway and PD-1, compositions and uses thereof - Google Patents

Bispecific antigen binding proteins targeting the VEGF pathway and PD-1, compositions and uses thereof Download PDF

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CN118620082A
CN118620082A CN202310230191.XA CN202310230191A CN118620082A CN 118620082 A CN118620082 A CN 118620082A CN 202310230191 A CN202310230191 A CN 202310230191A CN 118620082 A CN118620082 A CN 118620082A
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孙建明
管静
黄潇
房行行
李凤霞
凌虹
康小强
赖寿鹏
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Nanjing Wei Li Zhi Bo Biotechnology Co ltd
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Nanjing Wei Li Zhi Bo Biotechnology Co ltd
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Priority to PCT/CN2024/081040 priority patent/WO2024188222A1/en
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Abstract

The present invention provides bispecific antigen binding proteins targeting the VEGF pathway and PD-1, compositions and uses thereof. The invention also provides nucleic acid molecules, expression vectors and host cells encoding the bispecific antigen binding proteins. The invention also provides compositions and uses comprising the bispecific antigen binding proteins, and methods of treating diseases, particularly cancer, using the bispecific antigen binding proteins of the invention.

Description

Bispecific antigen binding proteins targeting the VEGF pathway and PD-1, compositions and uses thereof
Technical Field
The invention relates to the field of medicines, in particular to a bispecific antibody simultaneously targeting VEGF (vascular endothelial growth factor) pathway and PD-1, a preparation method, a pharmaceutical composition and application thereof.
Background
According to world health organization data, cancer is a major cause of death worldwide, and 1000 tens of thousands of people die from cancer in 2020. Efforts have been made to diagnose early and treat cancer correctly, and the overall mortality and number of people suffering from cancer are decreasing.
Cancer is in fact a group of diseases involving abnormal cell growth and division. Cancer cells are very "smart" and several mechanisms have been developed to evade immune surveillance of the host. For example, they express high levels of the membrane proteins PD-L1 and PD-L2, which bind to PD-1 on the surface of T cells and induce T cell apoptosis. Cancer cells also secrete angiogenic factors, such as Vascular Epithelial Growth Factor (VEGF), to promote the growth of new blood vessels into the tumor. With newly created blood vessels, cancer cells grow larger and faster, and thus have better shuttle capacity.
Current options for cancer treatment include surgery, radiation therapy, chemotherapy, hormonal therapy, targeted therapy, immunotherapy, and the like. Among them, antibody therapy has been rapidly developed, and has been quite successful in recent years. In general, antibodies can help the immune system kill cancer cells, prevent development of tumor vessels, or kill cancer cells directly.
PD-1, also known as CD279, is a cell surface receptor. It has two ligands, PD-L1 and PD-L2. The expression of PD-L1 on macrophages and dendritic cells is up-regulated in response to LPS and GM-CSF treatment, the expression of PD-L1 on T cells and B cells is up-regulated in T Cell Receptor (TCR) and B Cell Receptor (BCR) signaling, and PD-L1 mRNA is detectable in the heart, lung, thymus, spleen and kidney of mice. PD-L1 is also expressed on tumor cells. PD-L2 has a more restricted expression profile.
When bound to PD-L1 or PD-L2, PD-1 down regulates the immune system and promotes self-tolerance by inhibiting T cell inflammatory activity. The inhibition of the immune system by PD-1 may prevent autoimmune diseases. On the other hand, increased local PD-L1 expression in cancer cells may prevent the immune system from killing these cells. Several anti-PD-1 antibodies, e.g(BMS)(Merck) has been shown to be useful in clinical cancer therapy alone or in combination with other antineoplastic agents.
Vascular Endothelial Growth Factor (VEGF) is a signaling protein produced by cells that is involved in angiogenesis (vasculogenesis and angiogenesis). VEGF can be classified into VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF. VEGF overexpression may lead to disease. For example, cancers that express VEGF (e.g., VEGF-A and VEGF-D) are capable of growing and metastasizing under A sufficient blood supply. VEGF-A over-release was found to play A role in rheumatoid arthritis, diabetic retinopathy, wet age-related macular degeneration and glomerular hypertrophy.
Is a humanized anti-VEGF antibody that has been approved for the treatment of various cancers, including colorectal, lung and breast cancers.
VEGF receptors are natural receptors for VEGF, mainly three subtypes VEGFR-1, VEGFR-2 and VEGFR-3, each with a domain consisting of 7 immunoglobulins, a transmembrane region (a single transmembrane spanning region) and an intracellular portion.
Ramucirumab (Ramucirumab) is a human IgG1 monoclonal antibody that blocks vascular endothelial growth factor receptor 2 (VEGFR-2/VEGFR 2/KDR), thereby inhibiting angiogenesis. The FDA has approved for the treatment of gastric cancer, lung cancer and liver cancer.
Antibodies targeting a single tumor-associated antigen have been found to have limited therapeutic efficacy. For example, anti-VEGF antibodiesIt has been demonstrated that to some extent cancer cell growth is inhibited, but that cancer cells cannot be eliminated.
Thus, in clinical trials and clinical treatments, antibodies are typically administered in combination with another antibody or another therapeutic agent such that target cells, such as cancer cells, are challenged by the immune system in a variety of ways. Alternatively, single molecule drugs with two or more target binding specificities, such as bispecific or multispecific antibodies/proteins, can be developed to achieve therapies targeting more than one molecule. Currently available techniques for preparing bispecific or multispecific antibodies are Triomab, crossMab, DVD-Ig, biTE, DART and TandAb, among others. Bispecific antigen binding proteins (e.g., bispecific antibodies) of the same target exhibit different properties due to different structures, combinations of antibodies, and the like.
Disclosure of Invention
There is no currently marketed bispecific antigen binding protein-related drug targeting the VEGF pathway and PD-1, and therefore there is still a need for co-targeted drugs.
The bispecific antibody can specifically target VEGF pathway and PD-1, specifically relieve the immunosuppression of PD-1 on the organism, and inhibit angiogenesis caused by tumor by inhibiting the combination of VEGF and VEGFR, thereby having good application prospect.
The present invention provides a bispecific antigen binding protein comprising (a) a first antigen binding portion that specifically binds PD-1; and (b) a second antigen binding portion that specifically targets the VEGF pathway; and the second antigen binding portion comprises VEGFR1 domain 2 or comprises an antibody or antigen binding protein that targets VEGFR2
In some embodiments, the first antigen binding portion comprises: a heavy chain complementarity determining region and a light chain complementarity determining region comprising, in order from the N-terminus to the C-terminus: HCDR1, said HCDR1 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 1; HCDR2, said HCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 2; and HCDR3, said HCDR3 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 3; the light chain complementarity determining region comprises, in order from the N-terminus to the C-terminus: LCDR1, said LCDR1 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 4; LCDR2, said LCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 5; and LCDR3, said LCDR3 having an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 6;
The second antigen binding portion comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the heavy chain complementarity determining region comprising, in order from the N-terminus to the C-terminus: HCDR1, said HCDR1 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 7; HCDR2, said HCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 8; and HCDR3, said HCDR3 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 9; the light chain complementarity determining region comprises, in order from the N-terminus to the C-terminus: LCDR1, said LCDR1 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 10; LCDR2, said LCDR2 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 11; and LCDR3, said LCDR3 having an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 12; and the second antigen binding portion targets VEGFR2; or alternatively
The second antigen binding portion comprises VEGFR1 domain 2.
In some embodiments, the second antigen binding portion comprises the sequence set forth in SEQ ID NO. 20 for VEGFR1 domain 2.
In some embodiments, the N-terminus of the second antigen binding portion is linked to the C-terminus of the heavy chain of the first antigen binding portion by a first linker to form a recombinant Heavy Chain (HC). Preferably, the first connector is flexible. Preferably, the first linker is an IgG4 hinge region or (G 4S)n, where n is an integer from 1 to 5.
In some embodiments, wherein the bispecific antigen binding protein comprises a recombinant Heavy Chain (HC) comprising an anti-PD 1 antibody heavy chain, optionally a first linker, ramucirumab ScFv, and a Light Chain (LC); the Light Chain (LC) comprises an anti-PD 1 antibody light chain.
In some embodiments, wherein the bispecific antigen binding protein comprises a recombinant Heavy Chain (HC) comprising, in order from N-terminus to C-terminus, an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, optionally a first linker, a ramucirumab ScFv comprising a ramucirumab heavy chain variable region VH, a second linker, and a ramucirumab light chain variable region VL; the Light Chain (LC) comprises, in order from the N-terminus to the C-terminus, an anti-PD 1 antibody light chain variable region VL and an anti-PD 1 antibody light chain constant region CL. Preferably, the second connector is flexible. Preferably, the second linker may be the same as or different from the first linker.
In some embodiments, wherein the bispecific antigen binding protein comprises a recombinant Heavy Chain (HC) comprising, in order from N-terminus to C-terminus, an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, optionally a first linker, a heavy chain variable region VH of ramucirumab ScFv, a second linker, and a ramucirumab light chain variable region VL; the Light Chain (LC) comprises, in order from the N-terminus to the C-terminus, an anti-PD 1 antibody light chain variable region VL and an anti-PD 1 antibody light chain constant region CL. Preferably, the second connector is flexible. Preferably, the second linker may be the same as or different from the first linker.
In some embodiments, the anti-PD 1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 16; the IgG1 heavy chain constant region has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO. 17; the first linker is an IgG4 hinge region; (G 4S)n wherein n is an integer from 1 to 5, the heavy chain variable region VH of the ramucirumab ScFv has an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:18, the second linker is an IgG4 hinge region (G 4S)n wherein n is an integer from 1 to 5, preferably the second linker is (G 4S)n wherein n=3), the light chain variable region VL of the ramucirumab ScFv has an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:19, the anti-PD 1 antibody light chain variable region VL has an amino acid sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:21, and the anti-PD 1 antibody light chain constant region CL has an amino acid sequence at least 22%, at least 80%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.
In some embodiments, the recombinant Heavy Chain (HC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 13. Preferably, the recombinant Heavy Chain (HC) has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 13.
In some embodiments, the Light Chain (LC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 14. Preferably, the Light Chain (LC) has an amino acid sequence which is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 14.
In some embodiments, the second antigen binding portion is a ramucirumab ScFv, preferably the ramucirumab ScFv has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID No. 25.
In some embodiments, the bispecific antigen binding protein consists of two amino acid sequences set forth in SEQ ID NOs 13 and 14.
In some embodiments, the bispecific antigen binding protein comprises a recombinant Heavy Chain (HC) comprising, in order from N-terminus to C-terminus, an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional linker, VEGFR1 domain 2 (R1D 2), and a Light Chain (LC); the Light Chain (LC) comprises, in order from the N-terminus to the C-terminus, an anti-PD 1 light chain antibody variable region VL and a light chain constant region CL.
In some embodiments, the anti-PD 1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 16; the IgG1 heavy chain constant region has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO. 17; the first linker is an IgG4 hinge region; (G 4S)n), wherein n is an integer from 1 to 5, the VEGFR1 domain 2 (R1D 2) has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 20, the anti-PD 1 light chain antibody variable region VL has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 21, and the light chain constant region CL has an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 22.
In some embodiments, the recombinant Heavy Chain (HC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 15. Preferably, the recombinant Heavy Chain (HC) has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 15.
In some embodiments, the Light Chain (LC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 14. Preferably, the Light Chain (LC) has an amino acid sequence which is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO. 14.
In some embodiments, the bispecific antigen binding protein consists of the amino acid sequences set forth in SEQ ID NOs 15 and 14.
In some embodiments, the second antigen binding portion is VEGFR1 domain 2 (R1D 2). The invention also provides nucleic acid sequences encoding bispecific antigen binding proteins according to the foregoing.
In some embodiments, the HC comprises the following mutations: L234A, L235A, D265A, P329A.
The invention also provides a plasmid or vector comprising a nucleic acid sequence encoding the bispecific antigen binding protein described above, or comprising a nucleic acid sequence described above.
The invention also provides a host cell comprising and expressing the above nucleic acid sequence, plasmid or vector.
The present invention also provides a composition comprising a bispecific antigen binding protein according to the above.
The invention also relates to the use of the bispecific antigen binding proteins or compositions described above for the preparation of a medicament for the treatment of cancer.
The invention also provides a method of treating cancer comprising administering to a subject in need thereof the bispecific antigen binding protein or composition described above.
In some embodiments, preferred cancers for which bispecific antigen binding proteins or compositions comprising the same of the invention may be used to inhibit their growth include cancers that are generally responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include lung cancer, lymphoma, mesothelioma, melanoma, and renal cell carcinoma. In addition, the invention also includes refractory or recurrent malignancies whose growth can be inhibited using the bispecific antigen binding proteins of the invention or compositions comprising the same.
Examples of other cancers that may be treated using the bispecific antigen binding proteins of the invention or compositions comprising the same include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; childhood solid tumors, lymphocytic lymphomas, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphomas, environmentally induced cancers, including asbestos-induced cancers, and combinations thereof. The invention is also useful for the treatment of metastatic cancers, in particular those expressing PD-L1 (Iwai et al (2005) int. Immunol. 17:133-144).
Other features and advantages of the present application will become apparent from the following detailed description and examples, or may be learned by practice of the application, which are not to be construed as limiting. Other advantages of the application may be realized and attained by the structure particularly pointed out in the written description and drawings. The contents of all references, genbank entries, patents and published patent applications cited throughout this disclosure are expressly incorporated herein by reference.
Drawings
The accompanying drawings are included to provide an understanding of the principles of the application, and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain, without limitation, the principles of the application.
FIG. 1 shows the structure of a bispecific antigen binding protein of the invention, wherein R1D2 represents VEGFR1 domain 2.
FIG. 2 shows that the 1B12-R antibody can release the inhibition of NFAT signal by PD-1/PD-L1 binding.
FIG. 3 shows that the 1B12-R antibody can inhibit VEGF-A binding to VEGFR 2.
FIG. 4 shows that the 1B12-T antibody can release the inhibition of NFAT signal by PD-1/PD-L1 binding.
FIG. 5 shows that the 1B12-T antibody can inhibit VEGF-A binding to VEGFR 2.
FIG. 6 shows that the 1B12-T antibody inhibits VEGF-A induced proliferation of HUVEC cells.
FIG. 7 shows the in vivo anti-tumor effects of bispecific antigen binding proteins 1B12-R and 1B 12-T.
Detailed Description
In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term "and/or" as used herein shall be taken to be a specific disclosure of each of two or more specified features or components, with or without other features or components. Thus, the term "and/or" as used in the phrase, e.g., "a and/or B" herein, is intended to include "a and B", "a or B", "a" (alone) and "B" (alone).
It will be understood that wherever aspects are described by the language "comprising" or "including," similar aspects are also provided that are described by the terms "consisting of" and/or "consisting essentially of.
The term "about" means an amount, level, value, number, frequency, percentage, size, quantity, weight, or length that varies at an acceptable level in the art. In some embodiments, such variations may be up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference number, level, value, number, frequency, percentage, size, amount, weight, or length. When the term "about" is used in connection with a range of values, it modifies that range by extending the boundaries above and below the values.
The terms "antigen binding protein", "antibody" are used interchangeably and refer to a glycoprotein comprising at least two Heavy Chains (HC) and two Light Chains (LC) that are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant regions of antibodies may mediate binding of immunoglobulins to host tissues or factors.
The term "bispecific antigen binding protein" means a protein molecule having binding specificity for two different epitopes, wherein a first antigen binding portion and a second antigen binding portion can be linked together by, for example, a linker.
An antibody that "specifically binds to" human PD-1 as used herein is intended to refer to an antibody that binds to human PD-1 protein (and possibly PD-1 protein from one or more non-human species), but does not substantially bind to non-PD-1 protein.
The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a nucleotide sequence comprising deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule but is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".
The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may include mutations. Included herein are mutant progeny having the same function or biological activity as screened or selected in the original transformed cell.
IC50 (half maximal inhibitory concentration) refers to the half-inhibitory concentration of the antagonist being measured. It indicates that a certain drug or substance (inhibitor) is inhibiting half of a certain biological process (or a certain substance contained in the process, such as an enzyme, a cellular receptor or a microorganism). The IC50 measures the sensitivity of the antibody, the lower the IC50, the higher the sensitivity of the antibody.
EC50 (concentration for 50%of maximal effect) refers to the concentration that causes 50% of the maximum effect.
"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. The therapeutically effective amount of the antibody or antibody fragment or conjugate or composition thereof may vary depending on a variety of factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount of any toxic or detrimental effect of the antibody or antibody fragment or conjugate or composition thereof that is less than a therapeutically beneficial effect. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70% and still more preferably by at least about 80% or 90% relative to an untreated subject.
"Percent (%) sequence identity" with respect to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to residues in the reference sequence after aligning the candidate sequence to the reference sequence and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment used to determine the percent sequence identity can be accomplished in a variety of ways in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. When referring to percentages of sequence identity in the present application, these percentages are calculated relative to the full length of the longer sequence unless explicitly stated otherwise. The calculation of the full length relative to longer sequences applies to both nucleic acid sequences and polypeptide sequences.
The term "pharmaceutical composition" refers to a formulation in a form that is effective for the biological activity of the active ingredient contained therein and that is free of additional components that have unacceptable toxicity to the subject to whom the formulation is administered.
The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, and the like, as known in the art. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be pharmaceutically and physiologically acceptable in terms of compatibility with the other ingredients and not deleterious to the subject. Unless any conventional medium or agent is incompatible with the active ingredient, its administration in a therapeutic composition is contemplated.
It is to be understood that the term "treating" as used herein means reducing, preventing, curing, reversing, ameliorating, weakening, alleviating, minimizing, inhibiting or delaying the onset of one or more clinical indications of a disease or condition.
Examples
Example 1: construction, expression and purification of bispecific antibodies 1B12-R and 1B12-T of the invention
The invention constructs bispecific antibodies with two structures shown in figure 1, which are named as '1B 12-R' and '1B 12-T', wherein the anti-PD-1 antibody parts of the two bispecific antibodies are derived from the fully human anti-PD-1 antibody and from the PD1 antibody 21F12-1B12 in patent WO 2019219064A.
The heavy chain constant region of bispecific antibody is the heavy chain constant region of IgG1 with elimination effect function, which carries L234A, L235A, D265A, P329A, the amino acid sequence of which is ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:17).
The sequence from N end to C end of a recombinant Heavy Chain (HC) of the bispecific antibody 1B12-R is an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, a connector and ramucirumab ScFv, and the amino acid sequence of the recombinant Heavy Chain (HC) is SEQ ID NO. 13; the Light Chain (LC) of the bispecific antibody 1B12-R is an anti-PD 1 light chain antibody variable region VL and a light chain constant region CL in sequence from the N end to the C end, and the amino acid sequence of the Light Chain (LC) is SEQ ID NO. 14.
The sequence from N end to C end of the recombinant Heavy Chain (HC) of the bispecific antibody 1B12-T is an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, a connector and a VEGFR1 domain 2 (R1D 2), and the amino acid sequence of the recombinant Heavy Chain (HC) is SEQ ID NO. 15; the Light Chain (LC) of the bispecific antibody 1B12-T is an anti-PD 1 light chain antibody variable region VL and a light chain constant region CL in sequence from N end to C end, and the amino acid sequence of the Light Chain (LC) is SEQ ID NO. 14.
The nucleotides encoding the recombinant heavy and light chains of bispecific antibodies 1B12-T and 1B12-R were produced by gene synthesis (gold srey) and cloned into the expression vector pcdna3.1 (Invitrogen).
Expression vectors encoding the heavy and light chains described above were transfected into CHO-S cells, respectively, to express bispecific antibodies (ExpiCHO expression system, manufacturer: sieimer). Cells were cultured for 10-12 days after transfection, when the cell viability was reduced to 60% to 70%, the supernatant was collected, and antibodies expressed in the supernatant were purified using a MabSelect Sure protein a affinity chromatography system (GE HEALTHCARE) to obtain 1B12-R and 1B12-T. Concentrating the purified antibody, sterile filtering, detecting the purity of the antibody by SDS-PAGE and molecular exclusion, wherein the purity is more than 90%, and the antibody can be used for subsequent experiments.
TABLE 1 sequence composition of bispecific antibodies
Example 2: fortebio assay for affinity of 1B12-T for binding to human PD-1
In this experiment, the binding affinity of 1B12-T to human PD-1 (ACROBiosystems, cat#PD1-H5221) protein was tested using ForteBio Octet RED according to the manufacturer's instructions.
Briefly, an AHC sensor (forteBio, cat # 18-5060) was placed into Running Buffer (Running Buffer,1X PBS Hyclone,Cat#SH30256.01, containing 0.02% Tween20, pH 7.0) and pre-equilibrated for 10min at room temperature. In a 96-well plate, the kinetic assay was performed as follows: a) equilibration of baseline 100s with running buffer, B) addition of 1B12-T diluted with running buffer, final concentration 5. Mu.g/mL, solidification 200s, c) equilibration of baseline 300s with running buffer, d) addition of 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM of human PD-1 protein diluted with running buffer, binding 200s, dissociation 600s to each well. Experimental data were fitted and calculated using the Fortebio DATA ANALYSIS software 1:1 binding model. Table 1 summarizes the binding affinities of 1B12-T to human PD-1 proteins.
Table 2:1B12-T binding affinity with human PD1 protein
Antibodies to Analyte(s) Kon(M-1S-1) Koff(S-1) KD(M)
1B12-T Human PD1 proteins 1.30E+5 6.20E-4 4.75E-9
Example 3: fortebio assay for affinity of 1B12-T antibodies to bind human VEGF-A
In this experiment, the binding affinity of 1B12-T to human VEGF-A (SinoBiological, cat# 11066-HNAH) protein was tested using ForteBio Octet RED according to the manufacturer's instructions.
Briefly, an AHC sensor (forteBio, cat # 18-5060) was placed into running buffer (1X PBS Hyclone,Cat#SH30256.01, containing 0.02% Tween20, pH 7.0) and pre-equilibrated for 10min at room temperature. In a 96-well plate, the kinetic assay was performed as follows: a) equilibration of baseline 100s with running buffer, b) addition of bispecific antibody diluted with running buffer, final concentration 5 μg/mL, immobilization 200s, c) equilibration of baseline 300s with running buffer, d) addition of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM of human VEGF-A protein diluted with running buffer, binding 200s, dissociation 600s to each well. Experimental data were fitted and calculated using the Fortebio DATA ANALYSIS software 1:1 binding model. Table 2 summarizes the binding affinities of 1B12-T to human VEGF-A proteins.
Table 3:1B12-T binding affinity to human VEGF-A protein
Antibodies to Analyte(s) Kon(M-1S-1) Koff(S-1) KD(M)
1B12-T Human VEGF-A proteins 1.02E+6 <1.0E-7 <1.0E-12
Example 4:1B12-R antibodies abrogate the inhibition of PD-1/PD-L1
Construction of Jurkat-PD-1/NFAT cell line: NFAT-luc2P reporter element (Promega, cat#E8481) was transfected into Jurkat cells by Gene Pul Ser X Cell TM (BIO-RAD), and then the NFAT-luc2P polyclonal cell strain was selected with hygromycin B (Invitrogen, cat# 10687010), and stable and highly NFAT-luc2P expressing monoclonal cells Jurkat/NFAT-luc were obtained after limiting dilution of the polyclonal cells. Eukaryotic expression vector containing human PD1 gene is transfected into Jurkat/NFAT-luc monoclonal cell through Gene Pul Ser X Cell TM (BIO-RAD), then puromycin (Invitrogen Cat#A 1113802) is used for screening to obtain Jurkat-PD-1/NFAT polyclonal cell strain, and the monoclonal cell Jurkat-PD-1/NFAT which is stable and highly expresses human PD1 is obtained after limiting dilution of the polyclonal cell.
Construction of a CHO-K1/OKT3/PD-L1 cell line: eukaryotic expression vectors encoding CD3 antibodies (see amino acid sequence shown as SEQ ID NO:18 of China patent publication CN 114616251A) and human PDL1 (see amino acid sequence of NP-054862.1 shown as SEQ ID NO:17 of China patent publication CN 114616251A) were transfected into CHO-K1 cells using Lipofectamine TM 2000 (Invitrogen, cat# 11668019) transfection reagent according to the manufacturer's instructions, and 4. Mu.g/mL puromycin (Invitrogen, cat#A 1113802) and 600. Mu.g/mL neomycin (Invitrogen, cat# 10131027) were selected to give a polyclonal stable cell line of CHO-K1 expressing human PD-L1 and OKT3, and the polyclonal cells were subjected to limiting dilution to give stable and highly expressing monoclonal cells CHO-K1/OKT3/PD-L1 of human PD-L1 and OKT 3.
The Jurkat-PD-1/NFAT cell suspension was collected by centrifugation, the supernatant was discarded, the cell pellet was resuspended in assay medium (90% RPMI1640 medium+10% fetal bovine serum), counted in Countstar cell counter, and the cell suspension was diluted to 1X 10 6/mL with assay medium. The CHO-K1/OKT3/PD-L1 cells were collected by digestion centrifugation, the supernatant was discarded, the cell pellet was resuspended in assay medium, counted in Countstar cell counter, and the cell suspension was diluted to 5X 10 5/mL with assay medium. 5 Xfinal detection concentration of antibody to be tested was prepared. The final detection concentrations of the antibodies tested were 0.0002nM, 0.0061nM, 0.0024nM, 0.0098nM, 0.0391nM, 0.1563nM, 0.625nM, 2.5nM, 10nM and 40nM. Jurkat-PD-1/NFAT and CHO-K1/OKT3/PD-L1 cell suspensions were mixed at a volume ratio of 1:1, and the mixture was inoculated into 384 well assay plates, and 50. Mu.L/well was loaded. The gradient antibody dilutions were loaded at 10 μl/well. The whole plate is evenly mixed by a microplate rapid vibrator at 450rpm at room temperature for 5-10min, and is placed in a cell incubator with 37 ℃ and 5% CO 2 and saturated humidity for 6 hours. Thawing the One-Glo TM Buffer at room temperature, adding into the One-Glo TM Substrate, and fully dissolving at room temperature for later use. Taking out the plates to be tested from the incubator, balancing at room temperature for 10-15 min, adding 30 mu L of One-Glo TM Luciferase ASSAY REAGENT (Promega, catalog number: E6130) into each hole, uniformly mixing, cracking for 5-10 min at room temperature, using a fluorescence detection module of a multifunctional enzyme-labeled instrument (Tecan F200 Pro), selecting 384 Kong Baiban, and detecting the reading of a luminous signal. The GRAPHPAD PRISM software was entered and the x-axis data were Log of molar concentration, and the "Dose-response-Stim. Mu. Lation →log (agonist) vs. response-Variable slope (four parameters)" method was selected for fitting. EC 50 values were recorded.
As shown in FIG. 2, the 1B12-R antibody can release the inhibition of the NFAT signal by PD-1/PD-L1 binding, and has a dose-dependent trend in a reporter gene system, and the EC 50 value is 0.257nM.
Example 5:1B12-R antibodies inhibit VEGF-A binding to VEGFR2
Construction of 293T/NFAT/VEGFR2 cell lines: eukaryotic expression vectors encoding human VEGFR2 genes and NFAT-luc2P reporter elements (Promega, cat#E8481) were transfected into 293T cells using Lipofectamine TM 2000 (Invitrogen, cat# 11668019) transfection reagents according to manufacturer's instructions, and after 48 hours of transfection, were screened with 0.3. Mu.g/mL puromycin (Invitrogen, cat#A 1113802) and 85. Mu.g/mL hygromycin B (Invitrogen, cat# 10687010) to give 293T polyclonal stable cell lines that highly expressed human VEGFR2 and NFAT-luc2P, and the polyclonal cells were subjected to limiting dilution to give stable and highly expressing NFAT-luc2P and monoclonal cells Jurkat/NFAT/VEGFR2 of human VEGFR2.
The 293T/NFAT/VEGFR2 cells were collected by digestion centrifugation, the supernatant was discarded, the cell pellet was resuspended in assay medium (90% DMEM+10% fetal bovine serum), counted in Countstar cell counter, and the cell suspension was diluted to 5X 10 5/mL with assay medium. The 5 Xfinal detection concentration of the antibody to be tested was prepared with A measurement medium containing 15ng/mL VEGF-A. The final detection concentrations of the antibodies to be tested were 0.002nM, 0.0039nM, 0.0078nM, 0.0156nM, 0.0313nM, 0.0625nM, 0.125nM, 0.25nM, 0.5nM and 1nM. 293T/NFAT/VEGFR2 cell suspensions were seeded into 384 well plates, 40. Mu.L per well. The gradient antibody dilutions were loaded at 10 μl/well. The whole plate is evenly mixed by a microplate rapid vibrator at 450rpm at room temperature for 5-10min, and is placed in an incubator with 37 ℃ and 5% CO 2 and saturated humidity for 6 hours. Thawing the One-Glo TM Buffer at room temperature, adding into the One-Glo TM Substrate, and fully dissolving at room temperature for later use. Taking out the plates to be detected from the incubator, balancing for 10-15 min at room temperature, adding 30 mu LOne-Glo TM Luciferase ASSAY REAGENT into each hole, uniformly mixing, cracking for 5-10min at room temperature, using a fluorescence detection module of a multifunctional enzyme-labeled instrument, selecting 384-Kong Baiban, and detecting the reading of the luminous signals. The GRAPHPAD PRISM software was entered and the x-axis data were log of molar concentration, and the "Dose-response-Stimulation. Fwdarw. Log (inhibitor) vs. response-Variable slope (four parameters)" method was selected for fitting. EC 50 values were recorded.
The results of the experiment are shown in FIG. 3, in which the 1B12-R antibody inhibited VEGF-A binding to VEGFR2 and exhibited A dose dependent trend with an IC 50 value of 0.5105nM.
Example 6:1B12-T antibody releases the inhibition of PD-1/PD-L1
The Jurkat-PD-1/NFAT cell line and the CHO-K1/OKT3/PD-L1 cell line were constructed in the same manner as in reference example 4 to examine the inhibition of the 1B12-T antibody by PD-1/PD-L1. The final detection concentrations of the antibodies tested were 0.0012nM, 0.0049nM, 0.0195nM, 0.0781nM, 0.3125nM, 1.25nM, 5nM and 20nM. Microplates were incubated and luminescence signal readings were measured as described in example 4 and EC 50 values were recorded.
As shown in FIG. 4, the 1B12-T antibody can release the inhibition of the NFAT signal by PD-1/PD-L1 binding, and has a dose-dependent trend in a reporter gene system, and the EC 50 value is 0.1645nM.
Example 7:1B12-T antibodies inhibit VEGF-A binding to VEGFR2
The 293T/NFAT/VEGFR2 cell line was constructed as described in reference example 5 to detect binding of VEGF-A to VEGFR2 by the 1B12-T antibody. The final detection concentrations of the antibodies to be tested were 0.0410nM, 0.1024nM, 0.256nM, 0.64nM, 1.6nM, 4nM, 10nM and 25nM. Microplates were incubated and luminescence signal readings were measured as described in example 5. The IC 50 values were recorded. As shown in FIG. 5, the 1B12-T antibody can inhibit the binding of VEGF-A and VEGFR2, and has A dose-dependent trend in A reporter gene system, and the IC 50 value is 0.7267nM.
Example 8:1B12-T antibodies inhibit VEGF-A induced proliferation of Human Umbilical Vein Endothelial Cells (HUVECs)
HUVEC (manufacturer: ATCC, cat# CRL-1730 TM) cells were collected by digestion centrifugation, the supernatant was discarded and the cell pellet was resuspended in assay Medium (90% Medium-199 1X Earle's Salts (manufacturer: gibco cat# 11150-059) +10% fetal bovine serum (manufacturer Gibco cat# 10099-141) +10mM HEPES (manufacturer Gibco cat# 15630080) +100 units/mL penicillin 100. Mu.g/mL streptomycin (manufacturer Gibco cat# 10378016)), counted by a Countstar cell counter and the cell suspension was diluted to 5X 10 4/mL with assay Medium. 200ng/mL VEGF-A (manufacturer: kirsrui, cat# Z03073) working solution was prepared using assay medium, and an experimental control containing VEGF-A alone and no antibody was set. The test antibody was prepared in a final concentration of 4X in the measurement medium. The final detection concentrations of the antibodies tested were 0.1371nM, 0.4115nM, 1.2346nM, 3.7037nM, 11.1111nM, 33.3333nM, 100nM and 300nM. A96-well plate was inoculated with 100. Mu.L of HUVEC cell suspension, 50. Mu.L of VEGF-A working solution and 50. Mu.L of antibody. The 96-well plate was incubated in an incubator at 37℃with 5% CO 2 and saturated humidity for 96 hours. The culture supernatant was carefully aspirated prior to detection and 50 μl of fresh assay medium was added to each well. Cell viability was measured by adding 50. Mu. L CELL TITER Glo, reading the plates on a microplate reader F200 and recording the data. The GRAPHPAD PRISM software was entered and the x-axis data were log of molar concentration, and the "Dose-response-Stimulation. Fwdarw. Log (inhibitor) vs. response-Variable slope (four parameters)" method was selected for fitting. IC50 values were recorded.
The results of the experiment are shown in FIG. 6, in which the 1B12-T antibody inhibited VEGF-A induced proliferation of HUVEC cells and exhibited A dose-dependent trend with an IC50 of 4.370nM.
Example 9: in vivo anti-tumor effects of bispecific antibodies 1B12-R and 1B12-T
Humanized mouse C57BL/6JSmoc-Cd274 em1(hCD274)Kdrtm2(hKDR)Smoc expressing human PD-1/KDR was purchased from Shanghai, south mode Biotech Co., ltd.
HPDL 1A 1-B16F10 murine melanoma cell line was supplied by Shanghai, south mode Biotechnology Co., ltd. HPDL-B16F 10 cells were maintained in DMEM complete medium containing 10% fetal bovine serum. C57BL/6JSmoc-Cd274 em1(hCD274)Kdrtm2(hKDR)Smoc mice were subcutaneously implanted with 1×10 6 hPDL-B16F 10 cells and randomly divided into 4 groups (n=6 per group) when the average tumor volume reached about 70mm 3(LxW2/2 (day 0). Mice were administered 63.72nM/kg of 1B12-R (13 mg/kg), 1B12-T (11 mg/kg), 21F12-1F6 (9 mg/kg) and PBS intraperitoneally on days 0, 3, 7 and 10, respectively. 21F12-1F6 is an anti-PD-1 antibody 1B12 highly homologous antibody, the sequence of which is described in International patent application publication WO2019219064A. Tumor volumes were monitored by caliper measurements twice weekly during the experiment.
Treatment with 1B12-R and 1B12-T resulted in significant tumor growth inhibition compared to the PBS group, and 1B12-R and 1B12-T groups showed higher TGI rates (63.4% and 74% relative to 30.6%) compared to the anti-PD-1 antibody 21F12-1F6 group, as shown in Table 3 below.
Daily observations of the mice in each group showed no abnormalities; the weight of the mice is weighed twice a week, the weight of each group of mice is not obviously reduced, the weight of each group of mice at the end of the experiment is not obviously changed compared with the weight at the beginning of the administration treatment, and the drug has no obvious toxic or side effect on the mice.
TABLE 4 inhibition of hPDL1-B16F10 tumor growth
a Tumor volume data are expressed as mean ± SEM;
b Tgi= (tumor volume change of 1-administration group/tumor volume change of control group) ×100%
c Comparing with PBS group, performing two-way ANOVA, and performing Tukey's multiple comparison test
The present application has been described in terms of several embodiments, but the description is illustrative and not restrictive, and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the described embodiments. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are possible. Any feature of any embodiment may be used in combination with, or in place of, any other feature of any other embodiment, unless expressly limited otherwise.

Claims (20)

1. A bispecific antigen binding protein comprising (a) a first antigen binding portion that specifically binds PD-1; and (b) a second antigen binding portion that specifically targets the VEGF pathway; and the second antigen binding portion comprises VEGFR1 domain 2 or comprises an antibody or antigen binding protein that targets VEGFR 2.
2. The bispecific antigen binding protein of claim 1, wherein the first antigen binding moiety comprises: a heavy chain complementarity determining region and a light chain complementarity determining region comprising, in order from the N-terminus to the C-terminus: HCDR1, said HCDR1 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID No. 1; HCDR2, said HCDR2 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID No. 2; and HCDR3, said HCDR3 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID No. 3; the light chain complementarity determining region comprises, in order from the N-terminus to the C-terminus: LCDR1, said LCDR1 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 4; LCDR2, said LCDR2 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 5; and LCDR3, said LCDR3 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 6;
The second antigen binding portion comprises: a heavy chain complementarity determining region and a light chain complementarity determining region comprising, in order from the N-terminus to the C-terminus: HCDR1, said HCDR1 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID No. 7; HCDR2, said HCDR2 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID No. 8; and HCDR3, said HCDR3 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID No. 9; the light chain complementarity determining region comprises, in order from the N-terminus to the C-terminus: LCDR1, said LCDR1 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 10; LCDR2, said LCDR2 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 11; and LCDR3, said LCDR3 having an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 12; and the second antigen binding portion targets VEGFR2; or alternatively
The second antigen binding portion comprises VEGFR1 domain 2 (R1D 2).
3. The bispecific antigen binding protein of claim 2, wherein the N-terminus of the second antigen binding moiety is linked to the C-terminus of the heavy chain of the first antigen binding moiety by a first linker to form a recombinant Heavy Chain (HC); preferably, the first connector is flexible; preferably, the first linker is an IgG4 hinge region or (G 4S)n, where n is an integer from 1 to 5.
4. The bispecific antigen-binding protein of claim 2, wherein the bispecific antigen-binding protein comprises a recombinant Heavy Chain (HC) comprising, in order from N-terminus to C-terminus, an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional first linker, a ramucirumab ScFv comprising a ramucirumab heavy chain variable region VH, a second linker, and a ramucirumab light chain variable region VL; the Light Chain (LC) comprises, in order from the N-terminus to the C-terminus, an anti-PD 1 antibody light chain variable region VL and an anti-PD 1 antibody light chain constant region CL.
5. The bispecific antigen binding protein of claim 4, wherein
The anti-PD 1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75% identical to the amino acid sequence set out in SEQ ID NO. 16;
The IgG1 heavy chain constant region has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 17;
The first linker is an IgG4 hinge region; (G 4S)n, wherein n is an integer of 1 to 5;
The heavy chain variable region VH of the ramucirumab ScFv has an amino acid sequence which is at least 75% identical to the amino acid sequence listed in SEQ ID NO. 18;
The second linker is an IgG4 hinge region; (G 4S)n, wherein n is an integer from 1 to 5, preferably the second linker is (G 4S)n, wherein n=3;
The light chain variable region VL of the ramucirumab ScFv has an amino acid sequence which is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 19;
The anti-PD 1 antibody light chain variable region VL has an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 21; and
The anti-PD 1 antibody light chain constant region CL has an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 22.
6. The bispecific antigen binding protein of claim 5, wherein the recombinant Heavy Chain (HC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID No. 13.
7. The bispecific antigen binding protein of claim 6, wherein the Light Chain (LC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID No. 14.
8. The bispecific antigen binding protein of claim 7, wherein the second antigen binding moiety is a ramucirumab ScFv, preferably the ramucirumab ScFv has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID No. 25.
9. The bispecific antigen-binding protein of claim 2, wherein the bispecific antigen-binding protein comprises a recombinant Heavy Chain (HC) comprising, in order from N-terminus to C-terminus, an anti-PD 1 antibody heavy chain variable region VH, an IgG1 heavy chain constant region, an optional first linker, VEGFR1 domain 2 (R1D 2); the Light Chain (LC) comprises, in order from the N-terminus to the C-terminus, an anti-PD 1 light chain antibody variable region VL and a light chain constant region CL.
10. The bispecific antigen binding protein of claim 9, wherein
The anti-PD 1 antibody heavy chain variable region VH has an amino acid sequence that is at least 75% identical to the amino acid sequence set out in SEQ ID NO. 16;
The IgG1 heavy chain constant region has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 17;
The first linker is an IgG4 hinge region or (G 4S)n, wherein n is an integer from 1 to 5;
The VEGFR1 domain 2 (R1D 2) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 20;
The anti-PD 1 antibody light chain variable region VL has an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 21; and
The anti-PD 1 antibody light chain constant region CL has an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO. 22.
11. The bispecific antigen binding protein of claim 10, wherein the recombinant Heavy Chain (HC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID No. 15.
12. The bispecific antigen binding protein of claim 11, wherein the Light Chain (LC) has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID No. 14.
13. The bispecific antigen binding protein of any one of claims 3-12, wherein the HC comprises the following mutations: L234A, L235A, D265A, P329A.
14. A nucleic acid sequence encoding a bispecific antigen binding protein according to any one of claims 1-13.
15. A plasmid or vector comprising a nucleic acid sequence encoding a bispecific antigen binding protein according to any one of claims 1-13, or comprising a nucleic acid sequence according to claim 14.
16. A host cell comprising and expressing the nucleic acid sequence of claim 14 or the plasmid or vector of claim 15.
17. A composition comprising the bispecific antigen binding protein of any one of claims 1-13.
18. Use of a bispecific antigen binding protein according to any one of claims 1-13 or a composition according to claim 17 in the manufacture of a medicament for the treatment of cancer.
19. A method of treating cancer comprising administering to a subject in need thereof the bispecific antigen binding protein of any one of claims 1-13 or the composition of claim 17.
20. The use of claim 18 or the method of claim 19, wherein the cancer is: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia; childhood solid tumors, lymphocytic lymphomas, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphomas, environmentally induced cancers, including asbestos-induced cancers, and combinations thereof.
CN202310230191.XA 2023-03-10 2023-03-10 Bispecific antigen binding proteins targeting the VEGF pathway and PD-1, compositions and uses thereof Pending CN118620082A (en)

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US11136394B2 (en) * 2018-05-17 2021-10-05 Nanjing Leads Biolabs Co., Ltd. Antibody binding PD-1 and use thereof
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