A CD47 antibody

Abstract

The present application relates to a kind of CD47 antibodies, and the bispecific antibody comprising the CD47 antibody and VEGF antibody, the antibody molecule provided by the present application has high stability, easy to express and purify, has stronger tissue penetration, does not cause coagulation, does not bind red blood cell and cause antigen silencing effect, has excellent affinity to target point, can block relevant path, has high tumor killing efficiency, promotes tumor phagocytosis, and has multiple excellent effects such as strong, has good efficacy and safety.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to a CD47 antibody, a bispecific antibody comprising the CD47 antibody and a VEGF antibody, and their applications. Background Technology

[0002] CD47 (cell differentiation 47) is a highly glycosylated protein that spans five membranes and belongs to the immunoglobulin superfamily (IgSF). As a ubiquitously expressed protein, CD47 interacts with various extracellular ligands (such as integrins, SIRPα, and thrombolytic proteins) through its IgV domain, thereby participating in the regulation of different signaling pathways and playing an important role in the development and progression of diseases such as tumors, cardiovascular diseases, and autoimmune diseases. Cancer cells utilize the CD47-SIRPα-mediated "don't eat me" signal to upregulate CD47 expression in various tumors to achieve their escape. CD47 is one of the most promising immune checkpoint targets after PD1 / PDL1, but the development of antibody drugs targeting it faces numerous difficulties, and no related antibody drugs have been marketed to date. The most significant challenge is the safety of the drug. CD47 is also expressed in non-malignant cells of the hematopoietic system, including normal red blood cells, senescent red blood cells, and platelets. The binding of CD47 antibodies to blood cells can lead to coagulation or the death of normal red blood cells. Therefore, developing CD47 antibodies that bind weakly or not at all to blood cells, thereby solving the safety problem, is of paramount importance in developing related drugs to meet clinical needs.

[0003] Vascular endothelial growth factor (VEGF) is a highly specific pro-vascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. VEGF plays a crucial role in pathological angiogenesis, inducing the occurrence and progression of certain pathologies, such as tumor growth and metastasis, macular degeneration, diabetic retinopathy, inflammatory processes (e.g., rheumatoid arthritis), ischemic processes (myocardial ischemia), and preeclampsia. Vascular endothelial growth factor receptor (VEGFR) is a tyrosine kinase receptor that plays a key role in many signal transduction pathways essential for angiogenesis and cell migration. Upon binding to VEGF, it initiates a signaling cascade, thereby stimulating angiogenesis. Currently, blocking angiogenesis using anti-VEGF / VEGFR therapy is considered extremely important in cancer and other pathological processes. Inhibition of VEGF / VEGFR is a major treatment method for some cancers (e.g., renal cell carcinoma, hepatocellular carcinoma).

[0004] Compared to ordinary monospecific antibodies, bispecific antibodies have an additional specific antigen-binding site, allowing them to bind to two targets simultaneously, blocking two signaling pathways, reducing tumor escape, and improving treatment efficacy. Compared to combination therapies, bispecific antibodies are also significantly less expensive than combining two monotherapy drugs. However, bispecific antibodies also present significant development challenges, such as target sequence analysis and balancing and coordinating the safety and efficacy of the two targets. The development of CD47 and VEGF bispecific antibodies aims to achieve complementary and synergistic effects by modulating multiple pathways through tumor targets, angiogenesis mechanisms, and immune detection mechanisms, resulting in better patient outcomes. Furthermore, by evaluating antibody coagulation effects and binding to various cells, greater safety can be achieved. Current immunotherapy drugs and their rational application still have certain limitations and areas requiring improvement. Therefore, based on advances in basic medical and pharmaceutical research, further optimization and updates of immunotherapy drugs, as well as expanding the beneficiary population and improving efficacy, are key research directions that need to be addressed. Summary of the Invention

[0005] This invention relates to a molecule that specifically targets CD47, the molecule comprising 3 CDR regions and 4 FR regions.

[0006] Preferably, the molecule that specifically targets CD47 is VHH.

[0007] In one aspect, the CD47-targeting molecule contains the CDR1 amino acid sequence as shown in SEQ ID NO: 1, the CDR2 amino acid sequence as shown in SEQ ID NO: 2, and the CDR3 amino acid sequence as shown in SEQ ID NO: 3.

[0008] In one respect, the CD47-targeting molecule comprises:

[0009] (1) An FR1 amino acid sequence as shown in SEQ ID NO:4 or having 1-5 (including 1, 2, 3, 4 or 5) amino acid mutations compared to SEQ ID NO:4; and / or

[0010] (2) An FR2 amino acid sequence as shown in SEQ ID NO: 5 or having 1-3 (including 1, 2 or 3) amino acid mutations compared to SEQ ID NO: 5; and / or

[0011] (3) An FR3 amino acid sequence as shown in SEQ ID NO: 6 or having 1-8 (including 1, 2, 3, 4, 5, 6, 7 or 8) amino acid mutations compared to SEQ ID NO: 6; and / or

[0012] (4) An FR4 amino acid sequence as shown in SEQ ID NO:7 or having one amino acid mutation compared to SEQ ID NO:7.

[0013] Preferably, in the molecule targeting CD47:

[0014] (1) 1-5 (inclusive) mutations in FR1 are selected from mutations at positions 1, 5, 14, 21, 23, or combinations thereof; and / or

[0015] (2) One to three (inclusive) mutations in FR2 are selected from mutations at positions 43, 46, and 49, or combinations thereof; and / or

[0016] (3) Mutations 1-8 (including 1, 2, 3, 4, 5, 6, 7 or 8) in FR3 are selected from mutations at positions 58, 68, 72, 74, 78, 82B, 83, 84, or combinations thereof; and / or

[0017] (4) One mutation in FR4 is selected from the mutation at position 108.

[0018] Preferably, in the molecule targeting CD47:

[0019] (1) 1-5 (inclusive) mutations in FR1 are selected from Q1E, Q5V, A14P, A21V, T23A mutations, or combinations thereof; and / or

[0020] (2) One to three (including one, two, or three) mutations in FR2 are selected from N43K, D46E, A49S mutations, or combinations thereof; and / or

[0021] (3) 1-8 (including 1, 2, 3, 4, 5, 6, 7 or 8) mutations in FR3 are selected from D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A mutations, or combinations thereof; and / or

[0022] (3) One mutation in FR4 is selected from the Q108L mutation.

[0023] In one respect, the CD47-targeting molecule comprises:

[0024] (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 8; and / or

[0025] (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 9; and / or

[0026] (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 10; and / or

[0027] (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 11.

[0028] In one respect, the CD47-targeting molecule comprises:

[0029] (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4; and / or

[0030] (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5; and / or

[0031] (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6; and / or

[0032] (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7.

[0033] In one respect, the CD47-targeting molecule comprises:

[0034] (1) The FR1 amino acid sequence as shown in SEQ ID NO: 8; and / or

[0035] (2) The FR2 amino acid sequence as shown in SEQ ID NO: 9; and / or

[0036] (3) The FR3 amino acid sequence as shown in SEQ ID NO: 10; and / or

[0037] (4) The FR4 amino acid sequence as shown in SEQ ID NO: 11.

[0038] In one aspect, the CD47-targeting molecule comprises the amino acid sequence shown in SEQ ID NO: 12, or having 1-17 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) amino acid mutations compared to SEQ ID NO: 12, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12.

[0039] Preferably, the molecule targeting CD47 contains 1-17 mutations (including mutations at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) selected from mutations at positions 1, 5, 14, 21, 23, 43, 46, 49, 58, 68, 72, 74, 78, 82B, 83, 84, 108, or combinations thereof.

[0040] Preferably, the molecule targeting CD47 contains 1-17 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) mutations selected from Q1E, Q5V, A14P, A21V, T23A, N43K, D46E, A49S, D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A, Q108L mutations, or combinations thereof.

[0041] In one aspect, the CD47-targeting molecule comprises the amino acid sequence shown in SEQ ID NO: 13, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13.

[0042] This invention relates to the use of the above-mentioned molecules, VHH or their binding fragments in the construction of monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates or recombinant proteins.

[0043] This invention relates to monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, or recombinant proteins comprising the above-mentioned molecules, VHH, or their binding fragments.

[0044] This invention relates to a molecule that specifically targets CD47 and VEGF, comprising:

[0045] (a) Targeting the first binding domain of CD47;

[0046] (b) Targeting the second binding domain of VEGF.

[0047] On one hand, the first binding domain targeting CD47 includes three CDR regions and four FR regions.

[0048] In one aspect, the first binding domain targeting CD47 comprises the CDR1 amino acid sequence as shown in SEQ ID NO: 1, the CDR2 amino acid sequence as shown in SEQ ID NO: 2, and the CDR3 amino acid sequence as shown in SEQ ID NO: 3.

[0049] SEQ ID NO: 1: GISVSAND

[0050] SEQ ID NO: 2: ITGGGRT

[0051] SEQ ID NO: 3: WGATY

[0052] In one aspect, the first binding domain targeting CD47 includes:

[0053] (1) An FR1 amino acid sequence as shown in SEQ ID NO:4 or having 1-5 (inclusive: 1, 2, 3, 4 or 5) amino acid mutations compared to SEQ ID NO:4; and / or

[0054] (2) An FR2 amino acid sequence as shown in SEQ ID NO: 5 or having 1-3 (including 1, 2 or 3) amino acid mutations compared to SEQ ID NO: 5; and / or

[0055] (3) An FR3 amino acid sequence as shown in SEQ ID NO: 6 or having 1-8 (including 1, 2, 3, 4, 5, 6, 7 or 8) amino acid mutations compared to SEQ ID NO: 6; and / or

[0056] (4) An FR4 amino acid sequence as shown in SEQ ID NO:7 or having one amino acid mutation compared to SEQ ID NO:7.

[0057] Preferably, in the first binding domain of the target CD47:

[0058] (1) 1-5 (inclusive) mutations in FR1 are selected from mutations at positions 1, 5, 14, 21, 23, or combinations thereof; and / or

[0059] (2) One to three (inclusive) mutations in FR2 are selected from mutations at positions 43, 46, and 49, or combinations thereof; and / or

[0060] (3) Mutations 1-8 (including 1, 2, 3, 4, 5, 6, 7 or 8) in FR3 are selected from mutations at positions 58, 68, 72, 74, 78, 82B, 83, 84, or combinations thereof; and / or

[0061] (4) One mutation in FR4 is selected from the mutation at position 108.

[0062] Preferably, in the first binding domain of the target CD47:

[0063] (1) 1-5 (inclusive) mutations in FR1 are selected from Q1E, Q5V, A14P, A21V, T23A mutations, or combinations thereof; and / or

[0064] (2) One to three (including one, two, or three) mutations in FR2 are selected from N43K, D46E, A49S mutations, or combinations thereof; and / or

[0065] (3) 1-8 (including 1, 2, 3, 4, 5, 6, 7 or 8) mutations in FR3 are selected from D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A mutations, or combinations thereof; and / or

[0066] (4) One mutation in FR4 is selected from the Q108L mutation.

[0067] In one aspect, the first binding domain targeting CD47 includes:

[0068] (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 8; and / or

[0069] (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 9; and / or

[0070] (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 10; and / or

[0071] (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 11.

[0072] In one aspect, the first binding domain targeting CD47 includes:

[0073] (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4; and / or

[0074] (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5; and / or

[0075] (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6; and / or

[0076] (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7.

[0077] In one aspect, the first binding domain targeting CD47 includes:

[0078] (1) The FR1 amino acid sequence as shown in SEQ ID NO: 8; and / or

[0079] (2) The FR2 amino acid sequence as shown in SEQ ID NO: 9; and / or

[0080] (3) The FR3 amino acid sequence as shown in SEQ ID NO: 10; and / or

[0081] (4) The FR4 amino acid sequence as shown in SEQ ID NO: 11.

[0082] The sequence is as follows:

[0083] SEQ ID NO: 4: QVQLQESGGGLVQAGGSLRLACTAS

[0084] SEQ ID NO: 5: MRWYRQAPGNQRDLVAR

[0085] SEQ ID NO: 6: DYADSVKGRFAISRENAKNTVYLQMNGLKPEDTAVYYC

[0086] SEQ ID NO: 7: WGQGTQVTVSS

[0087] SEQ ID NO: 8: EVQLVESGGGLVQPGGSLRLVCAAS

[0088] SEQ ID NO: 9: MRWYRQAPGKQRELVSR

[0089] SEQ ID NO: 10: SYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

[0090] SEQ ID NO: 11: WGQGTLVTVSS

[0091] In one aspect, the first binding domain targeting CD47 comprises the amino acid sequence shown in SEQ ID NO: 12, or having 1-17 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) amino acid mutations compared to SEQ ID NO: 12, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity compared to SEQ ID NO: 12.

[0092] SEQ ID NO: 12:

[0093] QVQLQESGGGLVQAGGSLRLACTASGISVSANDMRWYRQAPGNQRDLVARITGGGRTDYADSVKGRFAISRENAKNTVYLQMNGLKPEDTAVYYCWGATYWGQGTQVTVSS

[0094] Preferably, the first binding domain targeting CD47 contains 1-17 mutations (including mutations at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) selected from mutations at positions 1, 5, 14, 21, 23, 43, 46, 49, 58, 68, 72, 74, 78, 82B, 83, 84, 108, or combinations thereof.

[0095] Preferably, the first binding domain targeting CD47 contains 1-17 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) mutations selected from Q1E, Q5V, A14P, A21V, T23A, N43K, D46E, A49S, D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A, Q108L mutations, or combinations thereof.

[0096] In one aspect, the first binding domain targeting CD47 comprises an amino acid sequence shown in SEQ ID NO: 13, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13.

[0097] SEQ ID NO: 13:

[0098] EVQLVESGGGLVQPGGSLRLVCAASGISVSANDMRWYRQAPGKQRELVSRITGGGRTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCWGATYWGQGTLVTVSS

[0099] In one aspect, the second binding domain targeting VEGF includes the CDR1 amino acid sequence as shown in SEQ ID NO: 14, the CDR2 amino acid sequence as shown in SEQ ID NO: 15, and the CDR3 amino acid sequence as shown in SEQ ID NO: 16.

[0100] SEQ ID NO: 14: GFTFSTST

[0101] SEQ ID NO: 15: ITSAGATT

[0102] SEQ ID NO: 16: RALVTLWNVY

[0103] In one aspect, the second binding domain targeting VEGF comprises an amino acid sequence shown in SEQ ID NO: 17, or having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.

[0104] SEQ ID NO: 17:

[0105] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVSFITSAGATTYYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCRALVTLWNVYWGQGTLVTVSS

[0106] In one respect, the molecules that specifically target CD47 and VEGF include:

[0107] (1) Contains a first binding domain targeting CD47 containing the amino acid sequence shown in SEQ ID NO: 13, and / or

[0108] (2) A second binding domain for targeting VEGF containing the amino acid sequence shown in SEQ ID NO: 17.

[0109] In one aspect, the first binding domain targeting CD47 and / or the second binding domain targeting VEGF are antibodies or their antigen-binding fragments.

[0110] Preferably, the first binding domain targeting CD47 and / or the second binding domain targeting VEGF are VHH.

[0111] In one aspect, the molecule specifically targeting CD47 and VEGF includes an FC domain. Preferably, the FC domain is selected from IgA, IgD, IgE, IgG, and IgM. Preferably, the FC domain is selected from IgG. Preferably, the FC domain is selected from IgG1 or IgG4. The hinge region included in the FC domain may be complete, partial, or missing.

[0112] In one aspect, the IgG4 FC domain comprises an amino acid sequence shown in SEQ ID NO: 18, or having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.

[0113] IgG4 FC amino acid sequence (SEQ ID NO: 18)

[0114] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0115] In one aspect, the VHH targeting CD47 is connected to the N-terminus of the FC domain via an optional peptide linker, and the VHH targeting VEGF is connected to the C-terminus of the FC domain via an optional peptide linker.

[0116] Preferably, the peptide linker is a flexible peptide linker; preferably, the amino acid sequence of the peptide linker is (GGGGS)n, where n is equal to 1, 2, 3 or 4.

[0117] In one respect, the molecule specifically targeting CD47 and VEGF contains the amino acid sequence shown in SEQ ID NO: 19, or having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.

[0118] SEQ ID NO: 19

[0119] EVQLVESGGGLVQPGGSLRLVCAASGISVSANDMRWYRQAPGKQRELVSRITGGGRTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCWGATYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG EVQLLESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYR QAPGKERELVSFITSAGATTYYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCRALVTLWNVYWGQGTLVTV SS

[0120] Note: Bold text indicates CD47#44 VHH; regular text indicates IgG4 FC (S228P); The underscore indicates VEGF#48 VHH

[0121] Preferably, the molecule specifically targeting CD47 and VEGF is a bispecific antibody targeting both CD47 and VEGF.

[0122] This invention relates to a polynucleotide that encodes any of the VHH, monospecific antibody, bispecific antibody, multispecific antibody, or recombinant protein described above.

[0123] This invention relates to a carrier comprising any of the polynucleotides described above.

[0124] This invention relates to a host cell, wherein the host cell comprises the vector described in any of the preceding claims, or wherein the genome of the host cell integrates the polynucleotides described in any of the preceding claims. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is *Escherichia coli* cells, etc. Preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell, etc.; preferably, the yeast cell is *Pichia pastoris* cells, etc. Preferably, the cell is a mammalian cell; preferably, the mammalian cell is a Chinese hamster ovary cell (CHO), human embryonic kidney cell (293), B cell, T cell, DC cell, or NK cell, etc.

[0125] This invention relates to a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments thereof described above (including but not limited to VHH, monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, recombinant proteins, etc.), polynucleotides, carriers, cells or combinations thereof, and pharmaceutically acceptable carriers thereof.

[0126] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, carriers, cells or combinations thereof described above in the preparation of medicaments for treating cancer.

[0127] This invention relates to a method for treating cancer, comprising administering to a subject in need an effective amount of any of the above-described antibodies or antigen-binding fragments thereof, polynucleotides, carriers, cells or combinations thereof.

[0128] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof described above for the treatment of cancer.

[0129] The present invention relates to antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof for the treatment of cancer.

[0130] This invention relates to a kit comprising any of the antibodies or antigen-binding fragments thereof described above, polynucleotides, vectors, cells or combinations thereof.

[0131] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof as described above in the preparation of diagnostic or detection kits.

[0132] This invention relates to a method for diagnosing or detecting cancer, comprising administering an antibody or antigen-binding fragment, polynucleotide, vector, cell, or combination thereof or a kit according to any one of the present invention to a subject or sample in need.

[0133] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof, or kits described above for the detection or diagnosis of cancer.

[0134] The VHH or bispecific antibodies provided by this invention have a variety of excellent effects, such as high stability, easy expression and purification, small molecular weight with stronger tissue permeability, no coagulation, no binding to red blood cells to cause antigen silencing effect, excellent affinity for target, ability to block related pathways, high tumor killing efficacy, and strong tumor phagocytosis promotion. They have good efficacy and safety. Attached Figure Description

[0135] Figure 1 Schematic diagram of the structure of a bispecific antibody.

[0136] Figure 2 A comparative study on the mechanisms by which CD47#44 and Lemzoparlimab analogs bind to erythrocytes.

[0137] Figure 3 Studies on the tendency of CD47#2, CD47#41-CD47#44, and Lemzoparlimab analogs to bind to tumor cells or red blood cells when erythrocytes and Raji cells are present simultaneously.

[0138] Figure 4 CD47#1-CD47#4 coagulation reaction study.

[0139] Figure 5 Study on coagulation reaction of CD47#41-CD47#44.

[0140] Figure 6 Study on coagulation reaction of CD47VEGF#2-CD47VEGF#5.

[0141] definition

[0142] Unless otherwise specifically stated otherwise, the practice of this invention will take place using conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the scope of the art, or in the same sense as commonly understood by one of ordinary skill in the art to which this invention pertains. Many of these are described below for illustrative purposes, and such techniques are well described in the literature.

[0143] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (such as any one of the three CDRs of an immunoglobulin light chain or any one of the three CDRs of an immunoglobulin heavy chain) and capable of specifically binding to an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, VHH, single-chain antibodies, chimeric antibodies, humanized antibodies, and humanized antibodies. The term "antibody" as used herein includes both naturally occurring and non-naturally occurring antibodies. Generally, naturally occurring antibodies (also called immunoglobulins) consist of two classes of polypeptide chains: a light chain and a heavy chain. The non-limiting antibody disclosed in this invention can be a complete tetraimmunoglobulin chain antibody composed of two heavy chains and two light chains. The heavy chain of an antibody can be any isoform, including IgM, IgG, IgE, IgA, or IgD, or subisoforms, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. Each heavy chain contains a variable domain (or variable region, VH) and a constant domain (or constant region, CH), which are linked together by disulfide bonds within the constant domain. Each light chain contains a variable domain (or variable region, VL) and a constant domain (or constant region, CL), which are each linked to one heavy chain by a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain it binds to. Both the light and heavy chain variable regions contain three hypervariable regions sandwiched between more conserved framework regions (FRs). These hypervariable regions, also known as complementarity-determining regions (CDRs), constitute the primary antigen-binding surface of the antibody. Determining the antibody's critical resistance (CDR) by analyzing its amino acid sequence is a well-known method, and many commonly used CDR definitions exist, including but not limited to IMGT, KABAT, CHOTHIA, ABM, and CONTACT. In some embodiments, the antibody described in this invention may be a VHH or a protein containing a VHH that has antigen-binding capabilities. In some embodiments, the antibody may contain a constant region or FC region of a human antibody. The heavy chain constant region or FC region may be derived from IgM, IgG, IgE, IgA, or IgD or their subisotypes, such as, but not limited to, IgG1, IgG2, IgG3, and IgG4. The light chain constant region may be derived from the kappa light chain or the lambda light chain. The term antibody also includes derivatives, such as adjustments, conversions, additions, or reductions in structural / functional fragments, such as antibody-drug conjugates.

[0144] As used herein, the term "antigen-binding fragment" refers to one or more protein domains (e.g., formed by amino acids of a single polypeptide, or by amino acids of two or more polypeptides (e.g., the same or different polypeptides)) capable of specifically binding to one or more of the same or different antigens (e.g., tumor antigens). In some examples, antigen-binding fragments can bind to antigens or epitopes with specificity and affinity similar to naturally occurring antibodies. In some embodiments, the antigen-binding fragment may be an antibody or a fragment thereof. The antigen-binding fragment comprises a portion of an antibody, preferably the antigen-binding region and / or variable region of the antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments. In some embodiments, the antigen-binding fragment may be VHH. In some embodiments, the antigen-binding fragment may include other optional structures. The antigen-binding fragments described herein are non-limiting examples, and other examples of antigen-binding fragments are known in the art.

[0145] The term "CDR" (complementarity-determining region) used herein refers to a hypervariable region, and the CDRs involved in this invention are defined using the IMGT system. Unless otherwise specified, the immunoglobulin residues involved in this invention are numbered using the Kabat index. The methods for defining CDRs using the IMGT system and for numbering amino acid residues using the Kabat index are well known to those skilled in the art, and can be used, for example, with tools available at "http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi" for annotation and analysis.

[0146] As used herein, the term "FC domain" generally includes a hinge region, a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain (i.e., Hinge-CH2-CH3). The hinge region can be complete, partial, or missing. Currently, the FC domain of immunoglobulin IgG is most commonly used for constructing fusion proteins or antibodies. Fusion with the FC domain increases the molecular weight, enhances the stability of the fusion molecule through FcRn-mediated recycling mechanisms, and prolongs its in vivo half-life. Simultaneously, the Fc domain can mediate various biological functions such as ADCC and CDC. Currently, IgG1 antibodies are the most widely used. In recent years, with the expansion of new indications and the emergence of antibody drugs with new mechanisms of action, IgG2 and IgG4 subtypes with lower cytotoxicity have gained attention. In this invention, the "FC domain" is preferably the Fc domain of an IgG1 antibody or the FC domain of an IgG4 antibody.

[0147] As used herein, the term "specificity" refers to an antigen-binding protein or antibody that selectively recognizes a specific epitope of an antigen. For example, natural antibodies are monospecific. As used herein, the terms "bispecific" or "multispecific" indicate that an antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen.

[0148] The “VHH domain” (variable domain of heavy chain of heavy-chain antibody), also known as VHH, VHH domain, VHH antibody, VHH antibody fragment, single-domain antibody, Nanobody, etc., originally came from the antigen-binding immunoglobulin variable domain of “heavy-chain antibody” (hcAb, i.e. “antibody lacking light chain”) (C. Hamers-Casterman, T. Atarhouch, et al. Naturally occurring antibodies devoid of light chains. Nature, June 3, 1993, Vol. 363, pp. 446-448). The term “VHH domain” is used to distinguish this type of variable domain from the heavy chain variable domain (VH or VH domain) present in conventional antibodies composed of two light chains and two heavy chains (hereinafter referred to as conventional antibodies) and the light chain variable domain (VL or VL domain) present in conventional antibodies. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional antibodies, where the VL and VH domains are combined to recognize epitopes). The VHH domain is an antigen recognition unit formed by a single immunoglobulin domain.

[0149] As used herein, the term "vector" refers to any construct capable of delivering one or more related polynucleotides to a host cell when introduced into the host cell. Vectors can be introduced into host cells using methods known in the art, such as electroporation, transfection, transformation, infection, and injection. Non-limiting examples of vectors include viral vectors, naked DNA or RNA, plasmids, etc. Based on vector properties, vectors can be classified as viral vectors (e.g., lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, bacteriophage vectors, etc.) and non-viral vectors (e.g., plasmids, etc.). Based on vector function, vectors can be classified as cloning vectors, expression vectors, etc. Based on the type of recipient cell into which the vector enters, vectors can be classified as eukaryotic vectors, prokaryotic vectors, shuttle vectors, etc.

[0150] As used herein, the term "identity" refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence after sequence alignment and the introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0151] As used in this article, “treatment” refers to a clinical intervention designed to alter the natural course of a treatment within an individual or cell during a clinicopathological process. Desired therapeutic effects include slowing disease progression, improving or alleviating disease states, and mitigating or improving prognosis. For example, reducing or eliminating one or more symptoms associated with the treated disease or condition (such as cancer, inflammation, or an autoimmune disease).

[0152] As used herein, the term "effective amount" refers to an amount or dose sufficient to produce a beneficial or intended effect, including preventing, slowing, delaying, or inhibiting the progression of a disease (such as cancer). In the case of cancer, the effective amounts of VHH, bispecific antibodies, multispecific antigen-binding constructs, pharmaceutical compositions, and immunoconjugates provided in this application may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably prevent) the metastasis of tumor cancer cells; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent. As understood in the clinical setting, the effective amount of a drug, compound, or pharmaceutical composition may be achieved with or without combination with another drug, compound, or pharmaceutical composition.

[0153] The terms “subject” and “patient” as used herein are used interchangeably throughout the specification and are used to describe animals (human or non-human) to which they are treated by the method according to the invention. “Subject” is preferably a mammal, including but not limited to humans, cattle, horses, felines, canines, rodents, or primates. In some embodiments, the subject is a human. Detailed Implementation

[0154] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0155] Example

[0156] Example 1: Development of CD47 VHH Targeting Screening

[0157] Example 1.1 Screening and Control Construction

[0158] After screening using alpaca immunoglobulin and phage libraries, four sequences were identified that could bind to hCD47. These four sequences were then synthesized and constructed into the pcDNA3.4 vector, where they were fused with the hIgG4 Fc fragment for expression. The corresponding protein samples were named CD47#1 to CD47#4.

[0159] One of the control antibodies used is a Lemzoparlimab analogue, whose heavy chain amino acid sequence is shown in SEQ ID NO: 20 and light chain amino acid sequence is shown in SEQ ID NO: 21.

[0160] SEQ ID NO: 20

[0161] EVQLVESGGGLVKPGGSLRLSCAASGLTFERAWMNWVRQAPGKGLEWVGRIKRKTDGETTDYAAPVKGRFSISRDDSKNTLYLQMNSLKTEDTAVYYCAGSNRAFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0162] SEQ ID NO: 21

[0163] DIVMTQSPDSLAVSLGERATINCKSSQSVLYAGNNRNYLAWYQQKPGQPPKLLINQASTRASGVPDRFSGSGSGTEFTLIISSLQAEDVAIYYCQQYYTPPLAFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0164] The second control antibody was constructed according to the sequence described in Example 1 (Design of Anti-CD47 / VEGF Bispecific Antibody Molecular Sequence) of CN110885377B specification. The heavy chain was selected from scheme A, that is, the amino acid sequence of the heavy chain of the bispecific antibody molecule is as shown in SEQ ID NO: 13 of the patent. The CD47 single-domain antibody is linked to the C-terminus of the bevacizumab heavy chain through linker (GGGGS)4. The light chain of the bispecific antibody molecule is the bevacizumab light chain shown in SEQ ID NO: 11 of the patent. The construct was named BMK58-uIgG1.

[0165] Example 1.2 Antibody Thermal Stability Analysis

[0166] Take 10 µg of protein sample, add 1 µL of 5x SYPRO Orange (ThermoFisher, S6651) protein fluorescent dye, and then add 1x PBS to make up the volume to 20 µL. On a real-time quantitative PCR instrument, heat the sample from 25 °C to 95 °C at a rate of 0.05 °C / min, collecting fluorescence signals during the process to form protein melting curves. Analyze the protein melting curves using Protein Thermal Shift Software 1.4 (ThermoFisher) and calculate their Tm values. The results show that the Tm value of CD47#1 is only 56.3 °C, while the other samples exhibit good thermal stability: CD47#2 has a Tm value of 62.2 °C, CD47#3 has a Tm value of 61.2 °C, and CD47#4 has a Tm value of 62.1 °C.

[0167] Example 1.3 Antibody cellular level CD47 binding analysis

[0168] To investigate the binding capacity of the sample at the cellular level, CHO-K1 CD47 cells were digested with 0.25% Trypsin EDTA (Gibco, 25200-056) and the cell concentration was adjusted to 2 x 10⁻⁶. 6Cells / mL: Add 100 µL to each well of a 96-well cell culture plate, centrifuge, and discard the supernatant. Serially dilute the protein sample (5-fold serial dilution, maximum concentration 100 nM, 8 concentration points): Add 100 µL of the serially diluted protein sample to each well to resuspend the cells, mix well, and incubate at 4°C for 1 hour. Wash twice with PBS, then add secondary antibody Alexa Fluor. ® 647 Goat anti-human IgG Fc (Jackson, 109-605-098) was mixed and incubated at 4°C for 45 min. The cells were washed twice with 200 µL PBS and then resuspended in 100 µL PBS. After filtration through nonwoven fabric, the MFI value was read using a flow cytometer (BD, FACS Lyric), and the binding EC50 value was calculated using a Graphpad four-parameter fitting curve. The results showed that CD47#1, CD47#3, and CD47#4 did not bind to CHO-K1 CD47 cells, while only CD47#2 bound to CHO-K1 CD47 cells (Table 1).

[0169] Table 1. Results of CD47 binding assay at the cellular level

[0170]

[0171] Example 1.4 Antibody blocking CD47 / SIRPα interaction

[0172] Dilute hSIRPα IgG1 Fc (ACRO, SIA-H52A8) to 2 µg / mL with coating buffer, add 100 µL / well to an ELISA plate (Corning, 9018), mix well, and incubate overnight at 4°C. Wash away excess coating buffer with a plate washer, add 200 µL of blocking buffer, and incubate at room temperature for 1 hour. Mix 50 µL of diluted protein sample (5-fold serial dilution, starting at 100 µg / mL, for a total of 8 concentration points) and 50 µL of 5 µg / mL hCD47 (his tag) (ACRO, CD7-H5227), add to the sample wells, and incubate at room temperature for 2 hours. Wash away unbound protein sample, add 100 µL of diluted secondary antibody Anti-his-HRP (GenScript, 20H002237), and incubate at room temperature for 1 hour. Substrate chromogenic solution (Thermo, 2023) was added, and after chromogenic development with TMB solution, stop solution was added. The absorbance at OD450 nm was read using a microplate reader, and the binding IC50 value was calculated using a GraphPad four-parameter fitting curve. The results showed that CD47#2 could block the interaction between CD47 and SIRPα (Table 2).

[0173] Table 2. Results of antibody-mediated blocking of CD47 / SIRPα interaction

[0174]

[0175] Example 1.5 Antibody coagulation reaction and erythrocyte binding assay

[0176] We assessed the blood safety of the candidate molecules in this project through coagulation reaction and erythrocyte binding assays.

[0177] Red blood cell suspensions from two different donors were centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The red blood cell concentration (v / v) was diluted to 4% with 1xPBS. Protein was diluted at a 3:1 ratio to eight concentration gradients, with the highest concentration being 100 µg / mL. 25 µL each of the diluted protein and red blood cells were added to 96-well round-bottom cell culture plates (Corning, 3799), gently mixed, and incubated at 37°C with 8% CO2 for 3 h in a Memert incubator (ICO240). Results showed that CD47#1-CD47#4 did not induce coagulation. Figure 4 ).

[0178] Red blood cells from the above sources were diluted to 0.25%, and the binding ability of the candidate molecules to CD47 on red blood cells was analyzed by FACS. The results showed that CD47#2 did not bind to red blood cells.

[0179] In summary, we selected CD47#2 as the final molecule and carried out humanization modification.

[0180] The amino acid sequence of CD47#2 VHH is shown below:

[0181] SEQ ID NO: 12 (The bolded and underlined areas are CDR1-3 (IMGT system))

[0182] QVQLQESGGGLVQAGGSLRLACTAS GISVSAND MRWYRQAPGNQRDLVAR ITGGGRT DYADSVKGRFAISRENAKNTVYLQMNGLKPEDTAVYYC WGATY WGQGTQVTVSS

[0183] Table 3. Categorization of amino acid sequence position (Kabat number) for CD47#2 VHH

[0184]

[0185] Example 2: Humanization and Optimization of the Initial CD47 VHH Sequence

[0186] To further improve the humanization and safety of CD47#2 VHH, four sequences were obtained through three rounds of sequence optimization and modification, using structural biology protein structure simulation. These sequences are numbered CD47#41, CD47#42, CD47#43, and CD47#44. All four sequences share the same CDR1, CDR2, and CDR3.

[0187] Example 2.1 Antibody thermal stability analysis

[0188] Regarding thermal stability, the Tm values ​​of the aforementioned modified antibodies are comparable to those of the camel-derived antibodies. Specifically, the Tm value of CD47#41 is 62.0℃, the Tm value of CD47#42 is 63.1℃, the Tm value of CD47#43 is 61.9℃, and the Tm value of CD47#44 is 63.1℃.

[0189] Example 2.2 Investigation of antibody coagulation reaction and binding to various cells

[0190] Further testing was conducted on the candidate molecule's coagulation response and binding to various normal cells. In the coagulation assay, blood from five different donor sources was tested, and the results showed that the modified molecule did not induce coagulation. Figure 5 ).

[0191] Regarding erythrocyte binding, the optimized candidate molecules showed weaker binding ability than CD47#2 in all five donors, with CD47#44 exhibiting the lowest erythrocyte binding signal and also weaker than the control antibody Lemzoparlimab analogue (Table 4). These four candidate molecules also showed similar binding trends in platelet cell binding (Table 5).

[0192] Table 4. Results of the erythrocyte binding assay

[0193]

[0194] Table 5. Results of platelet binding assay

[0195]

[0196] Since CD47 is also expressed on some immune cells, the binding ability of candidate molecules to PBMCs was also tested. FACS analysis showed that the binding of the modified molecules to PBMCs was weaker than both the pre-humanized molecules and the control antibody Lemzoparlimab analogue, with CD47#44 showing the weakest binding (Table 6). Furthermore, when erythrocytes and Raji cells were present simultaneously, the modified candidate molecules tended to bind to tumor cells rather than erythrocytes. The erythrocyte binding signal of CD47#41~CD47#44 was lower than that of the control antibody Lemzoparlimab analogue. Figure 3 ).

[0197] Table 6. Experimental results of candidate molecules binding to PBMCs

[0198]

[0199] In summary, CD47#44 was selected as the optimal molecule after engineering modification, and its function was further verified.

[0200] Example 2.3 Antibody and its ability to block SIRPα and SIRPγ

[0201] The blocking ability of CD47#44 against CD47 binding receptors SIRPα and SIRPγ was detected by ELISA. The results showed that CD47#44 had a stronger blocking ability against both CD47 receptors than the control antibody Lemzoparlimab analogue. The IC50 value against SIRPα was only about one-quarter of that against the control antibody, and the IC50 value against SIRPγ was only about one-third of that against the control antibody (Table 7).

[0202] Table 7. Experimental results of CD47#44 blocking SIRPα and SIRPγ

[0203]

[0204] Example 2.4 Analysis of antibody-induced macrophage phagocytic function

[0205] Meanwhile, the effect of CD47#44 on macrophage phagocytic function was investigated. In MKN-45 and SKOV3 cell systems, the macrophage phagocytic function mediated by CD47#44 was comparable to that of the control antibody Lemzoparlimab analogue. However, in the MDA-MB-231 cell system, the phagocytic promotion ability of CD47#44 was stronger than that of the control antibody Lemzoparlimab analogue.

[0206] Example 2.5 Comparative Analysis of Antibody-Red Blood Cell Binding Mechanisms

[0207] Further investigation was conducted to determine whether the binding mechanisms of CD47#44 and the control antibody Lemzoparlimab analog to erythrocytes were the same. According to the mechanism of action of Lemzoparlimab analogs, glycosylation modification on erythrocytes creates steric hindrance, preventing antibody binding. Therefore, after treating erythrocytes with a deglycosylation enzyme, the binding of deglycosylated and undeglycosylated erythrocytes to the antibody was detected by FACS. The results showed that the Lemzoparlimab analog had a higher affinity for deglycosylated erythrocytes, while the binding curves of CD47#44 to untreated and deglycosylated erythrocytes almost overlapped and were both lower than those of the control antibody. This indicates that the binding of CD47#44 to erythrocytes is not affected by glycosylation modification, demonstrating a different mechanism of action than Lemzoparlimab analogs. Figure 2 ).

[0208] CD47#2 VHH (SEQ ID NO: 12):

[0209] QVQLQESGGGLVQAGGSLRLACTASGISVSANDMRWYRQAPGNQRDLVARITGGGRTDYADSVKGRFAISRENAKNTVYLQMNGLKPEDTAVYYCWGATYWGQGTQVTVSS

[0210] CD47#44 VHH (SEQ ID NO: 13):

[0211] E VQL V ESGGGLVQ P GGSLRL V C A ASGISVSANDMRWYRQAPG K QR E LV S RITGGGRT S YADSVKGRF T ISR D N S KNT L YLQMN S L RA EDTAVYYCWGATYWGQGT L VTVSS

[0212] Table 8. Optimized amino acid mutation sites for CD47#44 VHH sequence (using Kabat numbers from Table 3)

[0213]

[0214] For the CD47#44 VHH sequence, the mutations Q1E+Q5V+A14P+A21V+T23A are located in the FR1 region, N43K+D46E+A49S are located in the FR2 region, D58S+A68T+E72D+A74S+V78L+G(82B)S+K83R+P84A are located in the FR3 region, and Q108L is located in the FR4 region.

[0215] Example 3: Selection of CD47-terminal antibody sequence for bispecific antibody

[0216] Example 3.1 Construction of Bispecific Antibody

[0217] according to Figure 1 Configuration 1, as shown, involved cloning the CD47 VHH and VEGF VHH sequences into a eukaryotic expression vector containing human IgG4 Fc. The Fc portion of the bispecific antibody used the Ser228Pro mutation, and the VEGF VHH portion used the VEGF#48 sequence. Based on the different sequences used in the CD47 VHH portion, four different bispecific antibody molecules were constructed, named CD47VEGF#2 (CD47 VHH using CD47#41), CD47VEGF#3 (CD47#42 VHH), CD47VEGF#4 (CD47#43 VHH), and CD47VEGF#5 (CD47#44 VHH). After sequencing identification, the correct plasmid was used for downstream transient transfection expression. All four molecules were expressed normally, and the sample purity was high.

[0218] The amino acid sequence of VEGF#48 is shown below:

[0219] SEQ ID NO: 17 (The bolded and underlined areas are CDR1-3 (IMGT system))

[0220] EVQLLESGGGLVQPGGSLRLSCAAS GFTFSTST MSWYRQAPGKERELVSF ITSAGATT YYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYC RALVTLWNVY WGQGTLVTVSS

[0221] The amino acid sequence of CD47VEGF#5 is shown below:

[0222] SEQ ID NO: 19

[0223] EVQLVESGGGLVQPGGSLRLVCAASGISVSANDMRWYRQAPGKQRELVSRITGGGRTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCWGATYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG EVQLLESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYR QAPGKERELVSFITSAGATTYYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCRALVTLWNVYWGQGTLVTV SS

[0224] Note: Bold text indicates CD47#44 VHH; regular text indicates IgG4 FC (S228P); The underscore indicates VEGF#48 VHH

[0225] Example 3.2 Thermal stability analysis of dual resistance

[0226] The protein melting curves were analyzed and their Tm values ​​were calculated. The results showed that the Tm values ​​of all samples were higher than 62℃.

[0227] Example 3.3 Double Antibody Hemagglutination Reaction Test

[0228] Red blood cell suspensions from five different donors were centrifuged and the supernatant was discarded. 25 µL each of diluted protein and red blood cells were added to cell culture plates, gently mixed, and incubated at 37°C. Agglutination was then observed and analyzed after photographing. Results showed that none of the four penicillin antibody samples or the negative control PBS group caused coagulation. Figure 6 ).

[0229] Example 3.4 Binding analysis of bispecific antibodies and PBMCs

[0230] Adjust the PBMC cell concentration by adding 100 µL to each well of a 96-well cell culture plate, centrifuging, and discarding the supernatant. Serially dilute the protein sample (4-fold serial dilution, maximum concentration 100 nM, 8 concentration points in total). Add 100 µL of the serially diluted protein sample to each well to resuspend the PBMC cells, mix well, and incubate at 4°C for 1 hour. Wash twice with PBS, then add the secondary antibody Alexa Fluor. ®647 Goat anti-human IgG Fc was mixed and incubated at 4°C for 45 min. The samples were washed twice with 200 µL PBS and then resuspended in 100 µL PBS. Analyzed using flow cytometry (BD, FACSLyric). Results showed that each bispecific antibody sample bound PBMCs with lower affinity than the control antibody (Table 9).

[0231] Table 9. Results of binding of bispecific antibodies to PBMC

[0232]

[0233] Example 3.5 Analysis of selective binding of bispecific antibodies to Raji cells / erythrocytes

[0234] Raji cells were stained, centrifuged, and the supernatant was discarded. The cells were washed twice with PBS and resuspended. The protein sample concentration was diluted to 300 nM with PBS. The cells were plated, and 50 µL each of the prepared Raji cells, red blood cells, and protein sample were added to each well. The plates were incubated at 4°C for 1 hour. After washing twice with PBS, the secondary antibody Alexa Fluor was added. ® 647 Goat anti-human IgG Fc was mixed and incubated at 4°C for 45 min. The sample was washed twice with 200 µL PBS, then resuspended in 100 µL PBS, filtered through nonwoven fabric, and analyzed by flow cytometry. The results showed that the four penicillin antibody samples exhibited strong selective binding signals to Raji cells, while the binding signal to erythrocytes was almost undetectable.

[0235] Example 3.6 Selective Binding Analysis of Bispecific Antibody to H292 Cells / Red Blood Cells

[0236] H292 cells were stained, centrifuged, and the supernatant was discarded. The cells were washed twice with PBS and resuspended. The CD47VEGF#5 protein sample was diluted to 300 nM with PBS. The cells were plated, and 50 µL each of the prepared H292 cells, red blood cells, and protein sample were added to each well. The plates were incubated at 4°C for 1 hour. After washing twice with PBS, the secondary antibody Alexa Fluor was added. ® 647 Goat anti-human IgG Fc was mixed and incubated at 4°C for 45 min. The mixture was washed twice with 200 µL PBS and then resuspended in 100 µL PBS. After filtration through non-woven fabric, the sample was analyzed by flow cytometry. The results showed that CD47VEGF#5 exhibited a strong H292 binding signal but did not bind to erythrocytes.

[0237] Example 3.7 Binding analysis of bispecific antibody to human CD47 ELISA

[0238] Dilute hCD47(ECD) (Sino Biological) to 0.5 µg / mL with coating buffer, add 100 µL / well to the microplate, mix well, and incubate overnight at 4°C. Wash away excess coating buffer with a plate washer, add 200 µL of blocking buffer, and incubate at room temperature for 1 hour. Wash away the blocking buffer, add 100 µL of diluted protein sample (4-fold serial dilution, starting at 200 nM, for a total of 8 concentrations), and incubate at room temperature for 2 hours. Wash away unbound protein sample, add 100 µL of diluted secondary antibody Anti-IgG (HRP), and incubate at room temperature for 1 hour. Add substrate chromogenic solution (Thermo, 2023), and after TMB chromogenic development, add stop solution. Read the absorbance at OD450 nm using a microplate reader. ELISA analysis showed that the EC50 of the four bispecific antibody samples binding to the extracellular domain of human CD47 was lower than that of the control antibody Lemzoparlimab analogue, and their affinity was more than twice that of the control antibody (Table 10).

[0239] Table 10. Binding results of bispecific antibodies to human CD47 ELISA

[0240]

[0241] Example 3.8 Double Antibody In Vitro Macrophage Phagocytosis Assay

[0242] H292 cells were stained (37℃, 20 min), centrifuged, and the supernatant was discarded. The cells were washed twice with PBS and resuspended, and macrophages were collected for later use. The protein sample to be analyzed was diluted to 300 nM and added to 96-well cell culture plates, with 50 µL of H292 cells, macrophages, and protein sample per well. The plates were incubated at 37℃ for 2 hours. 100 µL of 200-fold diluted APC Anti-Human CD14 Antibody (Biolegend, B329919) was added to each well, mixed, and incubated at 4℃ for 45 min. The cells were washed twice with 200 µL PBS and then resuspended with 100 µL PBS. After filtration through non-woven fabric, the cells were analyzed by flow cytometry (BD, FACSLyric). The results showed that the phagocytic promotion ability of the tested bispecific antibody sample was significantly stronger than that of the control antibody Lemzoparlimab analogue (Table 11).

[0243] Table 11. Results of macrophage phagocytosis promoted by dual antibody exogenous stimulation

[0244]

[0245] Example 3.9 ELISA Analysis of Bispecific Antibody Binding to Human VEGF165 / Human CD47

[0246] Dilute hVEGF165 (Sino Biological, 11066-HNAB) to 0.5 µg / mL with coating buffer, add 100 µL / well to a microplate (Corning, 9018), mix well, and incubate overnight at 4°C. Wash away excess coating buffer with a plate washer, add 200 µL of blocking buffer, and incubate at room temperature for 1 hour. Wash away the blocking buffer, add 100 µL of diluted CD47VEGF#5 protein sample (5-fold serial dilution, starting concentration 150 nM, 8 concentration points), and incubate at room temperature for 2 hours. Wash away unbound protein sample, add 100 µL of hCD47 his in each well, incubate at room temperature for 1 hour, add 100 µL of diluted secondary antibody Anti-his-HRP (GenScript, A00612), and incubate at room temperature for 1 hour. Add substrate chromogenic solution (Thermo, 2023), develop with TMB solution, and then add stop solution. The absorbance at OD450nm was read using a microplate reader. Analysis showed that CD47VEGF#5 could also bind to CD47 after binding to human VEGF165, with an EC50 of 0.98 nM.

[0247] Example 3.10 ELISA analysis of bispecific antibody binding to human CD47 / human VEGF165 sequentially

[0248] Add 100 μL of hCD47 his to each well of a microplate (Corning, 9018), mix well, and incubate overnight at 4°C. Wash away excess coating buffer with a plate washer, then add 200 μL of blocking buffer and incubate at room temperature for 1 hour. Wash away the blocking buffer, then add 100 μL of diluted CD47VEGF#5 protein sample (5-fold serial dilution, starting at 150 nM, 8 concentration points), and incubate at room temperature for 2 hours. Wash away unbound protein sample, then add 100 μL of biotin-labeled hVEGF165 (ACRO, VE5-H82Q0) to each well, and incubate at room temperature for 1 hour. Add 100 μL of diluted secondary antibody Streptavidin-HRP (Abcam, ab7403), and incubate at room temperature for 1 hour. Add substrate chromogenic solution (Thermo, 2023), and after TMB chromogenic development, add stop solution. Read the absorbance at OD450 nm using a microplate reader. The results showed that CD47VEGF#5 could also bind to VEGF165 after binding to human CD47, with EC50=1.07nM.

[0249] Example 3.11 Dual Antibody Blockade of Human VEGF165 Binding to VEGFR1 and VEGFR2

[0250] VEGFR1 and VEGFR2 D1-D3 were coated onto 96-well microplates, 100 µL per well, mixed thoroughly, and incubated overnight at 4°C. Excess coating buffer was washed away with a plate washer, and 200 µL of blocking buffer was added, followed by incubation at room temperature for 1 hour. The blocking buffer was washed away, and 100 µL of the mixed protein sample and hVEGF165 bio (ACRO, VE5-H82Q0) were added, followed by incubation at room temperature for 2 hours. Unbound protein sample was washed away, and 100 µL of diluted anti-SA-HRP secondary antibody (Abcam, ab7403) was added, followed by incubation at room temperature for 1 hour. Substrate chromogenic buffer (Thermo, 2023) was added, followed by TMB solution development, and then stop buffer was added. The absorbance at OD450 nm was read using a microplate reader. The results showed that CD47VEGF#5 could block the binding of human VEFG165 to VEGFR1 and also block the binding of VEFG165 to VEGFR2. Its IC50 value was smaller than that of the control antibody Bevacizumab analog (the heavy chain amino acid sequence of which is shown in SEQ ID NO: 22 and the light chain amino acid sequence of which is shown in SEQ ID NO: 23) and BMK58-uIgG1 bispecific antibody, indicating that CD47VEGF#5 has a stronger blocking ability on the VEFG165 signaling pathway (Table 12).

[0251] Table 12. Experimental results of dual-antibody blocking of human VEGF165 binding to VEGFR1 and VEGFR2

[0252]

[0253] SEQ ID NO: 22

[0254] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0255] SEQ ID NO: 23

[0256] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0257] Example 3.12 Bispecific Antibody Blocks Human VEGF165 / VEGFR2 Signaling Pathway

[0258] Collect HEK293 VEGFR2 NFAT cells and resuspend them in 5% FBS DMEM medium containing VEGF his. Adjust the cell concentration to 6x10 5Cells / mL. Add 50 µL of cells and diluted CD47VEGF#5 protein sample to a 96-well white plate and incubate at 37°C with 5% CO2 for 6 hours. Add 100 µL of luminescent reagent and read the fluorescence value using a microplate reader. The results showed that CD47VEGF#5 effectively blocked the VEGF165 / VEGFR2 signaling pathway at the cellular level, with an EC50 of 0.026 nM.

[0259] In summary, CD47#44 VHH, contained in CD47VEGF#5, was selected as the antibody sequence for the CD47 terminus.

[0260] The above description discloses only some embodiments of the present invention and is not intended to limit the present invention in any way. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described above. For those skilled in the art, various improvements and modifications can be made to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A molecule that specifically targets CD47, comprising the CDR1 amino acid sequence shown in SEQ ID NO: 1, the CDR2 amino acid sequence shown in SEQ ID NO: 2, and the CDR3 amino acid sequence shown in SEQ ID NO: 3; preferably, the molecule that specifically targets CD47 is VHH.

2. The molecule according to claim 1, wherein, The molecule also includes: (1) An FR1 amino acid sequence as shown in SEQ ID NO:4 or having 1-5 amino acid mutations compared to SEQ ID NO:4, wherein the 1-5 mutations in the FR1 are selected from mutations at positions 1, 5, 14, 21, 23, or combinations thereof; and / or (2) An FR2 amino acid sequence as shown in SEQ ID NO: 5 or having 1-3 amino acid mutations compared to SEQ ID NO: 5, wherein the 1-3 mutations in the FR2 are selected from mutations at positions 43, 46, 49, or combinations thereof; and / or (3) An FR3 amino acid sequence as shown in SEQ ID NO: 6 or having 1-8 amino acid mutations compared to SEQ ID NO: 6, wherein the 1-8 mutations in the FR3 are selected from mutations at positions 58, 68, 72, 74, 78, 82B, 83, 84, or combinations thereof; and / or (4) An FR4 amino acid sequence as shown in SEQ ID NO:7 or having one amino acid mutation compared to SEQ ID NO:7, wherein the one mutation in the FR4 is selected from the mutation at position 108.

3. The molecule according to claim 2, wherein: (1) The 1-5 mutations in FR1 are selected from Q1E, Q5V, A14P, A21V, T23A mutations, or combinations thereof; and / or (2) The 1-3 mutations in FR2 are selected from N43K, D46E, A49S mutations, or combinations thereof; and / or (3) The 1-8 mutations in FR3 are selected from D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A mutations, or combinations thereof; and / or (4) One of the mutations in FR4 is selected from the Q108L mutation.

4. The molecule according to claim 3, wherein, The molecule comprises: (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 8; and / or (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 9; and / or (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 10; and / or (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO:

11.

5. The molecule according to claim 4, wherein, The molecule comprises: A： (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4; and / or (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5; and / or (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6; and / or (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7; or B： (1) The FR1 amino acid sequence as shown in SEQ ID NO: 8; and / or (2) The FR2 amino acid sequence as shown in SEQ ID NO: 9; and / or (3) The FR3 amino acid sequence as shown in SEQ ID NO: 10; and / or (4) The FR4 amino acid sequence as shown in SEQ ID NO:

11.

6. The molecule according to claim 1, wherein the molecule comprises the amino acid sequence shown in SEQ ID NO: 12, or having 1-17 amino acid mutations compared to SEQ ID NO: 12, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

12.

7. The molecule according to claim 6, wherein, The molecule contains 1-17 mutations selected from mutations at positions 1, 5, 14, 21, 23, 43, 46, 49, 58, 68, 72, 74, 78, 82B, 83, 84, 108, or combinations thereof.

8. The molecule according to claim 7, wherein, The molecule contains 1-17 mutations selected from Q1E, Q5V, A14P, A21V, T23A, N43K, D46E, A49S, D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A, Q108L mutations, or combinations thereof.

9. The molecule according to claim 8, wherein the molecule comprises an amino acid sequence shown in SEQ ID NO: 13, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

13.

10. Use of any molecule according to claims 1-9 in the construction of monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, or recombinant proteins.

11. A monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein comprising any of the molecules described in claims 1-9.

12. A molecule that specifically targets CD47 and VEGF, comprising: (a) Targeting the first binding domain of CD47; (b) Targeting the second binding domain of VEGF; in: The first binding domain targeting CD47 comprises the CDR1 amino acid sequence as shown in SEQ ID NO: 1, the CDR2 amino acid sequence as shown in SEQ ID NO: 2, and the CDR3 amino acid sequence as shown in SEQ ID NO: 3; and / or The second binding domain targeting VEGF includes the CDR1 amino acid sequence as shown in SEQ ID NO: 14, the CDR2 amino acid sequence as shown in SEQ ID NO: 15, and the CDR3 amino acid sequence as shown in SEQ ID NO:

16.

13. The molecule according to claim 12, wherein, The first binding domain targeting CD47 further includes: (1) An FR1 amino acid sequence as shown in SEQ ID NO:4 or having 1-5 amino acid mutations compared to SEQ ID NO:4, wherein the 1-5 mutations in the FR1 are selected from mutations at positions 1, 5, 14, 21, 23, or combinations thereof; and / or (2) An FR2 amino acid sequence as shown in SEQ ID NO: 5 or having 1-3 amino acid mutations compared to SEQ ID NO: 5, wherein the 1-3 mutations in the FR2 are selected from mutations at positions 43, 46, 49, or combinations thereof; and / or (3) An FR3 amino acid sequence as shown in SEQ ID NO: 6 or having 1-8 amino acid mutations compared to SEQ ID NO: 6, wherein the 1-8 mutations in the FR3 are selected from mutations at positions 58, 68, 72, 74, 78, 82B, 83, 84, or combinations thereof; and / or (4) An FR4 amino acid sequence as shown in SEQ ID NO:7 or having one amino acid mutation compared to SEQ ID NO:7, wherein the one mutation in the FR4 is selected from the mutation at position 108.

14. The molecule according to claim 13, wherein: (1) The 1-5 mutations in FR1 are selected from Q1E, Q5V, A14P, A21V, T23A mutations, or combinations thereof; and / or (2) The 1-3 mutations in FR2 are selected from N43K, D46E, A49S mutations, or combinations thereof; and / or (3) The 1-8 mutations in FR3 are selected from D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A mutations, or combinations thereof; and / or (4) One of the mutations in FR4 is selected from the Q108L mutation.

15. The molecule according to claim 14, wherein, The first binding domain targeting CD47 includes: (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 8; and / or (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 9; and / or (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 10; and / or (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO:

11.

16. The molecule according to claim 15, wherein, The first binding domain targeting CD47 includes: A： (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4; and / or (2) The FR2 amino acid sequence as shown in SEQ ID NO: 5; and / or (3) The FR3 amino acid sequence as shown in SEQ ID NO: 6; and / or (4) The FR4 amino acid sequence as shown in SEQ ID NO: 7; or B： (1) The FR1 amino acid sequence as shown in SEQ ID NO: 8; and / or (2) The FR2 amino acid sequence as shown in SEQ ID NO: 9; and / or (3) The FR3 amino acid sequence as shown in SEQ ID NO: 10; and / or (4) The FR4 amino acid sequence as shown in SEQ ID NO:

11.

17. The molecule according to claim 13, wherein the first binding domain targeting CD47 comprises the amino acid sequence shown in SEQ ID NO: 12, or having 1-17 amino acid mutations compared to SEQ ID NO: 12, or having more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

12.

18. The molecule according to claim 17, wherein, The first binding domain targeting CD47 contains 1-17 mutations selected from mutations at positions 1, 5, 14, 21, 23, 43, 46, 49, 58, 68, 72, 74, 78, 82B, 83, 84, 108, or combinations thereof.

19. The molecule according to claim 18, wherein, The first binding domain targeting CD47 contains 1-17 mutations selected from Q1E, Q5V, A14P, A21V, T23A, N43K, D46E, A49S, D58S, A68T, E72D, A74S, V78L, G(82B)S, K83R, P84A, Q108L mutations, or combinations thereof.

20. The molecule according to claim 19, The first binding domain targeting CD47 comprises an amino acid sequence shown in SEQ ID NO: 13, or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with SEQ ID NO:

13. and / or The second binding domain targeting VEGF contains an amino acid sequence shown in SEQ ID NO: 17, or having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

17.

21. The molecule according to claim 20, wherein the molecule comprises an FC domain selected from IgA, IgD, IgE, IgG, and IgM.

22. The molecule according to claim 21, wherein, The FC domain is selected from IgG1 or IgG4, wherein the hinge region contained in the FC may be complete, partial or missing.

23. The molecule according to claim 22, wherein, The FC domain of the IgG4 contains an amino acid sequence that is identical to or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same as SEQ ID NO:

18.

24. The molecule according to claim 23, wherein, The VHH targeting CD47 is optionally linked to the N-terminus of the FC domain via a peptide linker, and the VHH targeting VEGF is optionally linked to the C-terminus of the FC domain via a peptide linker.

25. The molecule according to claim 24, wherein, The amino acid sequence of the peptide linker is (GGGGS)n, where n equals 1, 2, 3 or 4.

26. The molecule according to claim 25, wherein the molecule comprises an amino acid sequence shown in SEQ ID NO: 19, or having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

19.

27. A polynucleotide encoding the molecule of claims 1-9, 12-26 or the monospecific antibody, bispecific antibody, multispecific antibody or recombinant protein of claims 10-11.

28. A vector comprising the polynucleotide of claim 27.

29. A host cell comprising the vector of claim 28, or having the polynucleotide of claim 27 integrated into its genome, wherein the cell is a bacterial cell, a fungal cell, or a mammalian cell.

30. The cell according to claim 29, wherein the cell is an Escherichia coli cell, a Pichia pastoris cell, a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (293), a B cell, a T cell, a DC cell, or an NK cell.

31. A pharmaceutical composition comprising the molecules of claims 1-9, 12-26, or the monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein of claims 10-11, the polynucleotide of claim 27, the carrier of claim 28, the cell of claims 29-30, or a combination thereof, and a pharmaceutically acceptable carrier thereof.

32. Use of the molecules of claims 1-9, 12-26, or the monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate or recombinant protein of claims 10-11, the polynucleotide of claim 27, the carrier of claim 28, the cell of claims 29-30, or combinations thereof, in the preparation of a medicament for treating cancer.

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