Antibodies or antigen-binding fragments thereof targeting sirpα and their preparation and use

Abstract

The application discloses an antibody or antigen-binding fragment thereof targeting Sirp alpha, which comprises a light chain variable region and / or a heavy chain variable region, the antibody or antigen-binding fragment thereof binds human Sirp alpha-V1 and human Sirp alpha-V2, but weakly binds or does not bind human Sirp beta, Sirp gamma, and does not bind human T cells, and has the function of blocking the binding of Sirp alpha and CD47. The application also discloses a bispecific antibody comprising the same, a preparation method of the antibody or antigen-binding fragment thereof, and application thereof. The unique characteristics of the antibody or antigen-binding fragment thereof make it more suitable for the development of an antibody or antigen-binding fragment thereof for a human Sirp alpha target, and can be administered alone or in combination as a candidate drug, and in particular, provide a new and even better choice for combined immunotherapy of tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a Sirpα-targeting antibody or antigen-binding fragment thereof, a preparation method and application thereof, and further relates to a bispecific antibody comprising the Sirpα-targeting antibody or antigen-binding fragment thereof. BACKGROUND

[0002] SIRP family is a transmembrane glycoprotein belonging to immunoglobulin superfamily. According to the structure of transmembrane region and intracellular region, it is generally divided into Sirpα, Sirpβ and Sirpγ. The extracellular region of the three is highly homologous, and is composed of three Ig-like domains. The intracellular region of Sirpβ and Sirpγ is very short, only six and four amino acids respectively, and has no signal motif interacting with phosphatase. The four tyrosine residues in the cytoplasmic region of Sirpα (Signal regulatory protein α, also known as CD172α, SHPS-1, P84 or BIT) form two typical immune-receptor tyrosin-based inhibitory motifs (ITIM), which can be phosphorylated to accumulate, and then activated by the cytosolic protein tyrosine phosphatase connected with itself, so as to regulate the growth and activation of cells. In myeloid cells, Sirpα often acts as an inhibitory receptor, which inhibits the activation of immune cells by recruiting tyrosine phosphatase with SH2 domain. Sirpα was identified in the late 1990s, and its expression is on the membrane surface of myeloid cells including all types of macrophages, granulocytes, dendritic cells and nerve cells, and less on other cells. The gene encoding human Sirpα is a polymorphic gene, including various variants. The most common protein variants are Sirpα-V1 and Sirpα-V2 (NCBI sequence number NP_542970 (P78324) and CAA71403). The polymorphism in human Sirp leads to changes in the amino acids exposed on the surface, but does not affect the binding with CD47. In different ethnic groups, the expression levels of Sirpα protein variants are different. In the population of Europe, Africa, America and South Asia, the proportion of Sirpα-V2 high expression population is relatively low, accounting for only 8.6% in Africa; but in East Asian population, the proportion is as high as 41.3%. The existing anti-SIRPα antibodies are only for Sirpα-V1 high expression population, but 20% of the population in clinic is non-V1 form, and the existing anti-Sirpα antibodies cannot meet the needs of this part of patients. For example, the drug OSE-172 of Boehringer Ingelheim targeting Sirpα (see WO2017178653A2) is in clinical trial. OSE-172 is a monoclonal antibody targeting Sirpα expressed in cells of myeloid lineage, which only binds to human Sirpα-V1, but not to human Sirpα-V2, so it can only treat the patient population with high expression of Sirpα-V1 in drug development.In addition, OSE-172 does not bind to Sirpa of cynomolgus (cyno) origin (Cyno Sirpa), which brings inconvenience to the selection of primate cynomolgus in the preclinical safety evaluation study. In terms of selectivity to Sirpb, OSE-172 binds to Sirpb, and the selectivity is not good enough, and if it is applied in the process of clinical drug development, there may be side effects caused by off-target effects, which brings safety problems.

[0003] The interaction of CD47 and Sirpa was first discovered in 1999, and a large number of studies have confirmed that CD47 (also known as Integrin-associated protein, IAP) is widely expressed on the surface of normal cells, and by binding to Sirpa on the surface of macrophages, a “don’t eat me” signal is released, thereby protecting healthy cells from being “eaten” by macrophages. Cancer cells have also learned this mechanism: overexpressing CD47 on their surface, making macrophages treat them as “normal cells”, thereby escaping macrophage-mediated phagocytic attack. When Sirpa binds to CD47, it leads to receptor clustering, tyrosine phosphorylation and activation, and inhibits the accumulation of synaptophysin in macrophages. In this process, Sirp alpha with phosphorylated ITIM can recruit and activate tyrosine phosphatases SHP-1 and SHP-2, transmit inhibitory signals and inhibit the phagocytosis of macrophages, ultimately leading to immune escape of tumor cells. Therefore, blocking the binding of Sirpa and CD47 can restore the related functions of macrophages, and ultimately achieve the effect of treating tumors.

[0004] In addition, CD47 also binds to Sirpy (also known as SIRPg, SIRPgamma, CD172g or SIRP beta 2), another member of the SIRP family present on the surface of human T cells but not human myeloid cells. In contrast to the expression of SIRPa on myeloid cells, Sirpy is restrictedly expressed on T lymphocytes. Sirpy is not expressed in mice. Studies have shown that Sirpy-CD47 interaction mediates cell-cell adhesion, enhances superantigen-dependent T cell-mediated proliferation and co-stimulates T-cell activation (Piccio et al, Blood, 105:6, 2005).

[0005] Because of the high sequence homology between Sirpa and Sirpg, especially in the region that binds CD47, the anti-Sirpa antibodies disclosed in the prior art are also capable of binding Sirpg, and have adverse effects in humans, such as inhibiting the proliferation of T cells and reducing the immune response. Because the testing of the prior known antibodies was performed in mouse models that do not have the Sirpg gene, it was not possible to predict whether such side effects exist for the prior anti-CD47 antibodies or non-selective anti-Sirpa antibodies. For example, an antibody against Sirpa is disclosed in US20140242095A1, but it has relatively strong binding to Sirpb and Sirpg, and binds to human T cells, and if it is applied in the process of developing a clinical drug, there can be side effects caused by off-target effects, bringing safety problems.

[0006] Therefore, there is an urgent need in the art for antibodies that have better effects, such as a wider patient population (e.g., targeting both human populations expressing V1 and / or human populations expressing V2), are capable of binding Sirpa while not binding human Sirpb and Sirpg to reduce side effects, are capable of binding to Sirpa of primate origin to make preclinical studies more convenient, etc. SUMMARY

[0007] The present application provides an antibody or antigen-binding fragment thereof targeting Sirpα, a bispecific antibody, and a preparation method and application thereof, to overcome the defects in the prior art that antibodies cannot simultaneously bind to two forms of human Sirpα, Sirpα-V1 and Sirpα-V2, thus targeting more patient groups, cannot bind to human Sirpβ and Sirpγ while binding to Sirpα, thus reducing side effects, and the expression level is not high after humanization. Compared with the prior art, the antibody or antigen-binding fragment thereof of the present application has good Sirpα binding activity, can not only block the binding of human Sirpα (including Sirpα-V1 and Sirpα-V2) to human CD47, but also can be used as a new drug development to target Sirpα and CD47 binding to achieve the purpose of treating tumors. Moreover, the antibody or antigen-binding fragment thereof of the present application can simultaneously bind to two forms of human Sirpα, Sirpα-V1 and Sirpα-V2, and the binding activity is significantly higher than that of the prior art. Therefore, the antibody or antigen-binding fragment thereof of the present application has many advantages as a drug development, and can target more patient groups (human groups expressing Sirpα-V1 and / or human groups expressing Sirpα-V2). The antibody or antigen-binding fragment thereof of the present application does not bind to human Sirpβ and Sirpγ, nor to human T cells, so that the antibody or antigen-binding fragment thereof of the present application has better selectivity, avoiding the side effects caused by non-targeted action of T cell binding in the clinic. Preferably, the antibody or antigen-binding fragment thereof of the present application can also bind to cynomolgus (cyno) Sirpα (cyno Sirpα), especially to various polymorphic cyno Sirpα, so that the cynomolgus monkey (cynomolgus, cyno) can be selected in the preclinical safety evaluation study, bringing great convenience to preclinical pharmacology, toxicology and other studies. The results of post-translational modification (PTM) analysis show that the antibody or antigen-binding fragment thereof of the present application has the characteristics of low immunogenicity and small risk of drug development. The antibody or antigen-binding fragment thereof of the present application has a higher expression level after humanization, which provides convenience and saves cost for downstream production and process. In a preferred embodiment of the present application, the expression level of the humanized antibody of the present application is as high as 275 mg / L, which is about 4.5 times higher than that of the chimeric antibody and the antibody OSE-172 in the prior art. The bispecific antibody designed and screened based on the Sirpα antibody sequence of the present application can retain the functional activity of the bispecific antibody, and the binding activity to the two targets is close to that of the corresponding monoclonal antibody. Moreover, the change in the activity of blocking the binding of the antigen to the corresponding ligand is consistent with the change in the binding activity of the corresponding monoclonal antibody.These bispecific antibodies (referred to as SBody in the present application) have a similar structure to a conventional IgG, have a complete Fc as a normal antibody, and can be purified according to the normal antibody, so that the process is simple and has the advantage of low production cost. In summary, the unique properties of the antibody or antigen-binding fragment thereof of the present application make it more suitable for the development of antibodies or antigen-binding fragments thereof targeting the human Sirpα target, and can be administered alone or in combination as a candidate drug, especially providing a new and even better choice for combined PD-1 antibody immunotherapy for tumors.

[0008] To solve the above technical problems, the first aspect of the present application provides an antibody or antigen-binding fragment thereof targeting Sirpα, which comprises a light chain variable region, i.e. VL, and / or a heavy chain variable region, i.e. VH, binds human Sirpα-V1 and human Sirpα-V2, but weakly binds or does not bind human Sirpβ, Sirpγ, and does not bind human T cells, and has the function of blocking the binding of Sirpα and CD47. Therefore, the antibody or antigen-binding fragment thereof targeting Sirpα in the present application recognizes different epitopes from the antibodies of the prior art, and has more excellent technical effects than the prior art.

[0009] Preferably, the antibody or antigen-binding fragment thereof also binds one or more of cyno Sirpα L932, L933, L936 and L937, but does not bind cyno Sirpα L938 and L939; wherein the NCBI Reference Sequence number of the amino acid sequence of L932 is NP_001271679.1, the NCBI Reference Sequence number of the amino acid sequence of L933 is XP_015313155.1, the amino acid sequence of L936 is as shown in SEQ ID NO: 3, the amino acid sequence of L937 is as shown in SEQ ID NO: 4, the amino acid sequence of L938 is as shown in SEQ ID NO: 5, and the amino acid sequence of L939 is as shown in SEQ ID NO: 6.

[0010] In the present application, the "binding" of the antibody or antigen-binding fragment thereof to human Sirpα-V1 / human Sirpα-V2 and cyno Sirpα is generally "strong binding". The "strong binding" is generally EC 50less than 0.2 nM (more preferably less than 0.1 nM), which is defined mainly based on the experimental results in Table 5, Table 13, Table 6, Table 15, generally the binding ability of the antibody or antigen-binding fragment thereof to human Sirpα-V1 / human Sirpα-V2 is similar to or even better than that of Ref 1 to human Sirpα-V1 / human Sirpα-V2.

[0011] In the present application, unless otherwise specified, the "binding" refers to the EC 50 between 0.2 nM and 2 nM (i.e. 0.2 nM ≤ EC 50 <2 nM), the "weak binding" refers to the EC 50 between 2 nM and 10 nM (i.e. 2 nM ≤ EC 50 <10 nM), the "slight binding" generally refers to the EC 50 more than 10 nM and less than 50 nM (i.e. 10 nM ≤ EC 50 <50 nM). The "no binding" generally refers to the EC 50 ≥ 50 nM or no binding signal can be detected. This definition is mainly based on the experimental results in Table 15, i.e. the EC 50 value of Ref 1 binding to Sirpβ is about 0.126 nM, the EC 50 value of Ref 2 binding to Sirpβ is about 0.167 nM, the EC 50 value of Ref 1 binding to Sirpγ cannot be detected, and the EC 50 value of Ref 2 binding to Sirpγ is about 1.46 nM.

[0012] The values of the above-mentioned EC 50 are generally obtained by conventional experimental methods in the art, such as ELISA detection, and the values of the above-mentioned IC 50 are generally obtained by conventional blocking ELISA experiments.

[0013] More preferably, the VL comprises the following complementarity determining regions (CDRs) (defined according to the CCG numbering rule): a VL CDR1 as set forth in an amino acid sequence of SEQ ID NO: 11; a VL CDR2 as set forth in an amino acid sequence of SEQ ID NO: 12; and / or a VL CDR3 as set forth in an amino acid sequence of SEQ ID NO: 13; and / or,

[0014] the VH CDR1 as depicted in SEQ ID NO: 14; the VH CDR2 as depicted in SEQ ID NO: 15; and / or, the VH CDR3 as depicted in SEQ ID NO: 16; or, the VL has 3, 2, or 1 amino acid mutation(s) in the amino acid sequence of the VL CDR1, VL CDR2, VL CDR3, respectively, and / or, the VH has 3, 2, or 1 amino acid mutation(s) in the amino acid sequence of the VH CDR1, VH CDR2, VH CDR3, respectively.

[0015] In the similar "having 3, 2, or 1 amino acid mutation", the "amino acid mutation" refers to the mutation of amino acid in the sequence of the variant compared to the original amino acid sequence, including the insertion, deletion or substitution of amino acid based on the original amino acid sequence. An exemplary explanation is that the mutation of CDR can contain 3, 2 or 1 amino acid mutations, and the same or different number of amino acid residues between these CDRs can be optionally selected for mutation, for example, 1 amino acid mutation to CDR1, and no amino acid mutation to CDR2 and CDR3.

[0016] In the present application, the mutation can include the mutation known to the person skilled in the art at present, for example, some mutations that can be made to the antibody during the production or application of the antibody, for example, mutations to the site of PTM that can exist, especially in the CDR region, including mutations related to the aggregation of the antibody, asparagine deamidation (site (NG, NS or NH, etc.), aspartic acid isomerization (DG, DP) sensitive site, N glycosylation (N-{P}S / T) sensitive site and oxidation sensitive site, etc.

[0017] The CDR amino acid residues of the VL and VH regions of the antibodies or antigen binding fragments described herein are in number and position in accordance with the known Kabat, Contact, CCG, AbM, and Chothia, etc. numbering conventions. In the present application, the amino acid sequences of the CDRs listed above are shown according to the CCG definition convention (the sequences shown in the claims of the present application are also according to the CCG definition convention). However, it is well known in the art that the CDRs of an antibody can be defined in various ways in the art, such as the Kabat definition convention based on sequence variability (see, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, Bethesda, MD (1991)), and the Chothia definition convention based on the location of structural loop regions (see, J Mol Biol 273:927-48, 1997). The boundaries of a given CDR can vary depending on the scheme used to identify them, and the definition conventions described herein, and the CDR sequences defined for the antibodies described herein, are shown in Tables 7-12. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering used for the Kabat and Chothia schemes is based on the most commonly used antibody region sequence lengths, and is adapted for insertions (e.g., "30a") and deletions in some antibodies by the insertion of letters. The two schemes differ in the placement of certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Thus, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., a variable region) and individual CDRs of the antibody or region thereof (e.g., "VH CDR1, VH CDR2) are understood to encompass the complementarity determining regions as defined by any of the above known schemes described herein. While the scope of the claims of the present application is based on the sequences shown according to the CCG definition convention, the corresponding amino acid sequences according to other CDR definition conventions (e.g., the sequences corresponding to the different CDR numbering conventions listed below) should also fall within the scope of the present application.

[0018] For example, according to the definition of the Kabat numbering convention, the VL comprises CDRs as follows: VL CDR1 as set forth in the amino acid sequence of SEQ ID NO: 11; VL CDR2 as set forth in the amino acid sequence of SEQ ID NO: 12; and / or VL CDR3 as set forth in the amino acid sequence of SEQ ID NO: 13; and / or the VH comprises CDRs as follows: VH CDR1 as set forth in the amino acid sequence of SEQ ID NO: 17; VH CDR2 as set forth in the amino acid sequence of SEQ ID NO: 15; and / or VH CDR3 as set forth in the amino acid sequence of SEQ ID NO: 16.

[0019] For example, according to the definition of the AbM numbering convention, the VL comprises CDRs as follows: VL CDR1 as set forth in the amino acid sequence of SEQ ID NO: 11; VL CDR2 as set forth in the amino acid sequence of SEQ ID NO: 12; and / or VL CDR3 as set forth in the amino acid sequence of SEQ ID NO: 13; and / or the VH comprises CDRs as follows: VH CDR1 as set forth in the amino acid sequence of SEQ ID NO: 14; VH CDR2 as set forth in the amino acid sequence of SEQ ID NO: 18; and / or VH CDR3 as set forth in the amino acid sequence of SEQ ID NO: 16.

[0020] For example, according to the definition of the Chothia numbering convention, the VL comprises CDRs as follows: VL CDR1 as set forth in the amino acid sequence of SEQ ID NO: 11; VL CDR2 as set forth in the amino acid sequence of SEQ ID NO: 12; and / or VL CDR3 as set forth in the amino acid sequence of SEQ ID NO: 13; and / or the VH comprises CDRs as follows: VH CDR1 as set forth in the amino acid sequence of SEQ ID NO: 19; VH CDR2 as set forth in the amino acid sequence of SEQ ID NO: 20; and / or VH CDR3 as set forth in the amino acid sequence of SEQ ID NO: 16.

[0021] For example, according to the Contact Number Rule definition, the VL comprises the CDRs of: VL CDR1 as set forth in the amino acid sequence of SEQ ID NO: 21; VL CDR2 as set forth in the amino acid sequence of SEQ ID NO: 22; and / or, VL CDR3 as set forth in the amino acid sequence of SEQ ID NO: 23; and / or, the VH comprises the CDRs of: VH CDR1 as set forth in the amino acid sequence of SEQ ID NO: 24; VH CDR2 as set forth in the amino acid sequence of SEQ ID NO: 25; and / or, VH CDR3 as set forth in the amino acid sequence of SEQ ID NO: 26.

[0022] Preferably, the antibody targeting Sirpa is a murine antibody.

[0023] More preferably, the VL of the murine antibody is the amino acid sequence as set forth in SEQ ID NO: 9 or a mutation thereof; and / or, the VH of the murine antibody is the amino acid sequence as set forth in SEQ ID NO: 10 or a mutation thereof; more preferably, the VL of the murine antibody is encoded by the nucleotide as set forth in SEQ ID NO: 7; and / or, the VH of the murine antibody is encoded by the nucleotide as set forth in SEQ ID NO: 8.

[0024] The mutation is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the VL and / or VH, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the VL and / or VH, and maintains or improves the binding of the antibody to Sirpa; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably, at least 95% sequence identity; most preferably, at least 99% sequence identity.

[0025] Preferably, the antibody targeting Sirpa or the antigen binding fragment thereof further comprises a murine antibody constant region or a human antibody constant region; the murine antibody constant region comprises a heavy chain constant region and a kappa or lambda type light chain constant region of murine IgGl, IgG2a, IgG2b3 or IgG3, and the human antibody constant region comprises a heavy chain constant region and a kappa or lambda type light chain constant region of human IgGl, IgG2, IgG3 or IgG4.

[0026] More preferably, when the antibody targeting Sirpa or the antigen binding fragment thereof comprises the variable region of a murine antibody and a human antibody constant region, the human antibody constant region comprises a heavy chain constant region and a kappa type light chain constant region of human IgG4 with the amino acid sequences as set forth in SEQ ID NO: 28 and SEQ ID NO: 27, respectively.

[0027] Preferably, the antibody targeting Sirpa is a humanized antibody.

[0028] More preferably, the framework region of the humanized antibody comprises a human antibody heavy chain framework region and a human antibody light chain framework region;

[0029] Further more preferably, the human antibody light chain framework region is selected from one or more of 1) IGKV1-27*01, IGKV1-33*01, IGKV1-39*01, IGKV1-NL1*01, IGKV1 / OR10-1*01, IGKV1D-33*01, IGKV1D-39*01, IGKV1-12*01, IGKV1-12*02 and IGKV1-17*02, and the J gene is selected from one or more of 2) hJk1, hJk2.1, hJk2.2, hJk2.3, hJk2.4, hJk3, hJk4.1, hJk4.2 or hJk5, or a back mutation thereof; and / or, the human antibody heavy chain framework region is selected from one or more of 1) IGHV1-46*01, IGHV1-46*02, IGHV1-46*03, IGHV1-69*02, IGHV1-69*04, IGHV1-69*06, IGHV1-69*08, IGHV1-69*09, IGHV1-69*10 or IGHV1-69*14, and the J gene is selected from one or more of 2) hJh1, hJh2, hJh3.1, hJh3.2, hJh4.1, hJh4.2, hJh4.3, hJh5.1, hJh5.2, hJh6.1, hJh6.2 or hJh6.3, or a back mutation thereof; the number of amino acid sites of the back mutation is preferably 0-10.

[0030] Most preferably, the VL of the humanized antibody comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-34 or a mutation thereof; and / or, the VH sequence of the humanized antibody comprises an amino acid sequence as shown in any one of SEQ ID NOs: 35-41 or a mutation thereof; the mutation is a deletion, substitution or addition of one or more amino acid residues on the amino acid sequence of the VL and / or VH, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of the VL and / or VH, and maintains or improves the binding of the antibody or antigen binding fragment thereof to Sirpa; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most preferably at least 99% sequence identity.

[0031] In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 29; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 37. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 38. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 39. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 40. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 41. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 32; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 34; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36. In a preferred embodiment of the present application, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 39.In a preferred embodiment of the present application, the light chain variable region VL comprises an amino acid sequence as set forth in SEQ ID NO: 32; and the heavy chain variable region VH comprises an amino acid sequence as set forth in SEQ ID NO: 39. In a preferred embodiment of the present application, the light chain variable region VL comprises an amino acid sequence as set forth in SEQ ID NO: 33; and the heavy chain variable region VH comprises an amino acid sequence as set forth in SEQ ID NO: 39. In a preferred embodiment of the present application, the light chain variable region VL comprises an amino acid sequence as set forth in SEQ ID NO: 34; and the heavy chain variable region VH comprises an amino acid sequence as set forth in SEQ ID NO: 39. In a preferred embodiment of the present application, the light chain variable region VL comprises an amino acid sequence as set forth in SEQ ID NO: 29; and the heavy chain variable region VH comprises an amino acid sequence as set forth in SEQ ID NO: 39.

[0032] Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a human antibody kappa or lambda type light chain constant region or a mutation thereof; and / or, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgGl, IgG2, IgG3 and IgG4 or a mutation thereof.

[0033] More preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a human antibody kappa type light chain constant region.

[0034] More preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4.

[0035] Further more preferably, the light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48 or a mutation thereof; and / or, the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 43 or a mutation thereof.

[0036] In a preferred embodiment of the present application, the antibody or antigen-binding fragment thereof targeting Sirpa comprises a light chain as set forth in an amino acid sequence of SEQ ID NO: 42 and a heavy chain as set forth in an amino acid sequence of SEQ ID NO: 43.

[0037] In a preferred embodiment of the present application, the antibody or antigen-binding fragment thereof targeting Sirpa comprises a light chain as set forth in an amino acid sequence of SEQ ID NO: 44 and a heavy chain as set forth in an amino acid sequence of SEQ ID NO: 43. In a preferred embodiment of the present application, the antibody or antigen-binding fragment thereof targeting Sirpa comprises a light chain as set forth in an amino acid sequence of SEQ ID NO: 44 and a heavy chain as set forth in an amino acid sequence of SEQ ID NO: 43.

[0038] In a preferred embodiment of the present application, the Sirpα-targeting antibody or antigen-binding fragment thereof comprises a light chain as shown in the amino acid sequence of SEQ ID NO: 45 and a heavy chain as shown in the amino acid sequence of SEQ ID NO: 43.

[0039] In a preferred embodiment of the present application, the Sirpα-targeting antibody or antigen-binding fragment thereof comprises a light chain as shown in the amino acid sequence of SEQ ID NO: 46 and a heavy chain as shown in the amino acid sequence of SEQ ID NO: 43.

[0040] In a preferred embodiment of the present application, the Sirpα-targeting antibody or antigen-binding fragment thereof comprises a light chain as shown in the amino acid sequence of SEQ ID NO: 47 and a heavy chain as shown in the amino acid sequence of SEQ ID NO: 43.

[0041] In a preferred embodiment of the present application, the Sirpα-targeting antibody or antigen-binding fragment thereof comprises a light chain as shown in the amino acid sequence of SEQ ID NO: 48 and a heavy chain as shown in the amino acid sequence of SEQ ID NO: 43.

[0042] Preferably, the Sirpα-targeting antibody or antigen-binding fragment thereof includes an immunoglobulin, Fab, Fab', F(ab')2, Fv or single-chain Fv fragment (scFv), bispecific antibody, multispecific antibody, single-domain antibody, single-region antibody or any other antibody retaining the partial ability of the antibody to specifically bind to an antigen, or a monoclonal antibody or polyclonal antibody prepared from the above-mentioned antibodies. The monoclonal antibody can be prepared by various approaches and techniques, including hybridoma technology, phage display technology, single-lymphocyte gene cloning technology, etc., and the mainstream is to prepare a monoclonal antibody from a wild-type or transgenic mouse by hybridoma technology.

[0043] To solve the above technical problems, the second aspect of the present application provides a bispecific antibody comprising a first protein functional region and a second protein functional region, wherein the first protein functional region is a Sirpα-targeting antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and the second protein functional region is an antibody or antigen-binding fragment thereof targeting a non-Sirpα antigen. The bispecific antibody can retain the binding activity and functional activity of a single Sirpα antibody, and also maintain the binding and functional activity of the other protein functional region. Moreover, the bispecific antibody has a structure similar to that of a normal IgG antibody, and can be expressed and purified by conventional antibody expression and purification methods, and is stable.

[0044] The bispecific antibody of the present application can be a sequence-based IgG like bispecific antibody (SBody). These bispecific antibody molecules have a complete Fc as normal antibodies, so that the purification process can be carried out according to normal antibodies, and thus the process is simple and has the advantage of low production cost.

[0045] In a specific embodiment of the present application, in the bispecific antibody as described above, the non-Sirpα antigen is an immune checkpoint antigen or a tumor treatment target, the immune checkpoint antigen preferably includes PD-1, PD-L1, Tim3, or LAG3, and the tumor treatment target preferably includes CLDN18.2 (claudin 18.2); more preferably, the second protein functional region is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Tim3 antibody, an anti-LAG3 antibody, or an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; most preferably, the anti-PD-1 antibody is Nivolumab (Nivo) or Pembrolizumab (Pem), and the anti-PD-L1 antibody is Atezolumab, Avelumab, or Durvalumab.

[0046] In order to design a bispecific antibody with a simple production process and effective activity, the bispecific antibody of the present application is in the form of a normal IgG-like structure, and specifically, the light chain and / or heavy chain variable region of the protein functional region capable of targeting two target points is designed in the structure, and the two protein functional regions share the same heavy chain Fc region. Preferably, the antibody molecule of one target point is connected to one end of the light chain or heavy chain of the complete antibody of another target point in the form of one or more scFvs. In this way, it is not only possible to avoid the unevenness of the expression product caused by the expression of different heavy chain Fc and / or different light chains, such as the co-expression of Fc in the form of Knob and Fc in the form of Hole, which will have uneven Fc-Fc pairing forms during the expression process, bringing many inconveniences to the purification process; it is also possible to avoid the influence of the design of the cross of the light chain and the heavy chain partial region on the structure activity, as well as the Fc mismatch phenomenon in the process. Through the design of one or more scFvs, the activity against a specific target point can also be adjusted. Through the screening of different designs, the SBody with the optimal activity can be obtained. The obtained preferred SBody has the advantages of retaining the dual target point activity and drugability due to the differences in sequence and design, so that it can be used for drug development as a normal antibody.

[0047] In some embodiments, the bispecific antibody as described above, the first protein functional region is an immunoglobulin, and the second protein functional region is one or more, preferably two, scFv; or, the second protein functional region is an immunoglobulin, and the first protein functional region is one or more, preferably two, scFv; wherein the scFv comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are connected by a linker, the linker is preferably (Gly-Gly-Gly-Gly-Ser) w [hereinafter referred to as (G4S) w ]; the scFv is connected to the immunoglobulin by a linker, the linker is selected from the group consisting of a peptide segment commonly used in the art or (G4S) w ; the w is preferably an integer between 0 and 10, more preferably 1, 2, 3 or 4.

[0048] The bispecific design is summarized (general formula 1) as shown in Table 17 of the present application. In Table 17, the sequence containing the light chain means that the sequence can include the scFv connected to the light chain sequence in addition to the light chain sequence; the sequence containing the heavy chain means that the sequence can include the scFv connected to the heavy chain sequence in addition to the heavy chain sequence. Among them, T1 represents the first protein functional region against target 1 (such as Sirpa), and T2 represents the second protein functional region against target 2 (non-Sirpa). T1 (scFv) represents the scFv sequence of the antibody against target 1; T2 (scFv) represents the scFv sequence against target 2.

[0049] (scFv) n1 , (scFv) n2 , (scFv) n3 , (scFv) n4wherein n1, n2, n3, n4 are natural numbers, which can be 0, 1, 2, 3, etc., and in specific embodiments of the present application, at least one of n1, n2, n3, n4 is 1, and the rest are 0. VL represents the antibody light chain variable region sequence against target 1 or 2; VH represents the antibody heavy chain variable region sequence against target 1 or 2. LC represents the constant region sequence of light chain (kappa or lambda), preferably the human light chain constant region sequence; HC represents the heavy chain, including the constant region sequence of IgG1, IgG2, IgG3, IgG4 (abbreviated as HC-IgG1, HC-IgG2, HC-IgG3, HC-IgG4), preferably the human heavy chain constant region sequence (HC-hIgG). When the C-terminal of the heavy chain constant region is connected to the scFv or other protein sequence, the C-terminal last amino acid K can be mutated, preferably mutated to A. Thus, in Scheme 1, T1 is an immunoglobulin, and T2 is an scFv; in Scheme 2, T2 is an immunoglobulin, and T1 is an scFv; the scFv targets the same target; in Schemes 3 and 4, the scFv at both ends targets two different targets.

[0050] In Table 17, the scFv is a light chain variable region-linker-heavy chain variable region, and the N-terminal of the light chain variable region or the C-terminal of the heavy chain variable region is connected to the C-terminal or N-terminal of the light chain and / or heavy chain of the immunoglobulin via a linker, respectively; or the scFv is a heavy chain variable region-linker-light chain variable region, and the N-terminal of the heavy chain variable region or the C-terminal of the light chain variable region is connected to the C-terminal or N-terminal of the light chain and / or heavy chain of the immunoglobulin via a linker.

[0051] It should be noted that when the scFv is a light chain variable region-linker-heavy chain variable region, the C-terminal of the light chain variable region is connected to the linker, and the N-terminal of the heavy chain variable region is connected to the linker, thereby exposing the N-terminal of the light chain variable region and the C-terminal of the heavy chain variable region of the scFv, so that they can be connected to the light chain and / or heavy chain of the immunoglobulin via a linker. In the present application, when it is connected to the light chain of the immunoglobulin, the C-terminal of the heavy chain variable region of the scFv is preferably used to connect to the N-terminal of the heavy chain of the immunoglobulin via a linker in some specific embodiments; when it is connected to the heavy chain of the immunoglobulin, the N-terminal of the light chain variable region of the scFv is preferably used to connect to the C-terminal of the heavy chain of the immunoglobulin.

[0052] When the scFv is a heavy chain variable region-linker-light chain variable region, it is connected in such a way that the N-terminus of the light chain variable region is connected to the linker, which is then connected to the C-terminus of the heavy chain variable region, thus exposing the C-terminus of the scFv light chain variable region and the N-terminus of the heavy chain variable region, which can be connected to the light chain and / or heavy chain of the immunoglobulin through the linker. In this case, when it is connected to the light chain of the immunoglobulin, in some specific embodiments, it is preferred to use the C-terminus of the light chain variable region of the scFv to connect to the N-terminus of the immunoglobulin heavy chain; when it is connected to the heavy chain of the immunoglobulin, in some specific embodiments, it is preferred to use the N-terminus of the heavy chain variable region of the scFv to connect to the C-terminus of the immunoglobulin heavy chain. Preferably, the two scFvs are symmetrically connected to the C-terminus and / or N-terminus of the light chain and / or heavy chain of the immunoglobulin.

[0053] In the present application, the sequences of the antibodies for each target point involved in the bispecific design, except for the anti-Sirpa antibody sequences or antigen binding fragments thereof described in the present application, are from already published antibody sequences. These include anti-PD-1 antibodies Nivolumab / Opidivo (referred to as Nivo) and Pembrolizumab / Keytruda (referred to as Pem). The sequences of Nivolumab and Pembrolizumab can be found in public resources such as www.drugbank.ca.

[0054] More preferably, the bispecific antibody can be: the first protein functional region is scFv, and the second protein functional region is immunoglobulin; wherein the scFv of the first protein functional region comprises VL and VH as described in the first aspect of the present application.

[0055] In a preferred embodiment of the present application, the VL of the scFv of the first protein functional region comprises an amino acid sequence as set forth in SEQ ID NO: 29; and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35. In a preferred embodiment of the present application, the VL of the scFv of the first protein functional region comprises an amino acid sequence as set forth in SEQ ID NO: 30; and the VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 36-41. In a preferred embodiment of the present application, the VL of the scFv of the first protein functional region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 31-34; and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36. In a preferred embodiment of the present application, the VL of the scFv of the first protein functional region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 29 or 31-34; and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 39.

[0056] Preferably, the immunoglobulin comprises the amino acid sequences of the light chain variable region of Pem, the light chain constant region of a kappa chain, the heavy chain variable region of Pem, and the heavy chain constant region of hlgG4; or, the immunoglobulin comprises the amino acid sequences of the light chain variable region of Nivo, the light chain constant region of a kappa chain, the heavy chain variable region of Nivo, and the heavy chain constant region of hlgG4.

[0057] The C-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin via a linker; and the light chain variable region of the scFv is a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39; or,

[0058] The C-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin via a linker; and the light chain variable region of the scFv is a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39; or,

[0059] The C-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin via a linker; and the light chain variable region of the scFv is a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39; or,

[0060] The N-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the C-terminus of the two light chains of the immunoglobulin through a linker; and the light chain variable region of the scFv is a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 39.

[0061] In addition, the bispecific antibody can also include the following structure, the first protein functional region is an immunoglobulin, the immunoglobulin comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 48 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 43; and the second protein functional region is an scFv:

[0062] The sequence of the light chain variable region of the scFv is the light chain variable region of Pem, and the heavy chain variable region of the scFv is the heavy chain variable region of Pem.

[0063] More preferably, the bispecific antibody comprises the following light chain amino acid sequence and heavy chain amino acid sequence: a light chain amino acid sequence as shown in SEQ ID NO: 50 and a heavy chain amino acid sequence as shown in SEQ ID NO: 51; or a light chain amino acid sequence as shown in SEQ ID NO: 52 and a heavy chain amino acid sequence as shown in SEQ ID NO: 53; or a light chain amino acid sequence as shown in SEQ ID NO: 54 and a heavy chain amino acid sequence as shown in SEQ ID NO: 51; or a light chain amino acid sequence as shown in SEQ ID NO: 52 and a heavy chain amino acid sequence as shown in SEQ ID NO: 55; or a light chain amino acid sequence as shown in SEQ ID NO: 56 and a heavy chain amino acid sequence as shown in SEQ ID NO: 57.

[0064] To solve the above technical problems, the third aspect of the present application provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof targeting Sirpα as described in the first aspect of the present application or the bispecific antibody as described in the second aspect of the present application.

[0065] The preparation method of the nucleic acid is a conventional preparation method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof by artificial full-sequence synthesis method.

[0066] The skilled person in the art knows that the base sequence encoding the amino acid sequence of the above antibody or antigen-binding fragment thereof can be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a homolog of a polynucleotide. The homolog of the polynucleotide in the present application can be prepared by substituting, deleting or adding one or more bases of the sequence gene encoding the antibody or antigen-binding fragment thereof within the range of maintaining the activity of the antibody.

[0067] To solve the above technical problem, the fourth aspect of the present application provides a recombinant expression vector comprising the isolated nucleic acid according to the third aspect of the present application.

[0068] The recombinant expression vector can be obtained by conventional methods in the art, i.e. by linking the nucleic acid molecule according to the present application to various expression vectors. The expression vector can be any conventional expression vector in the art as long as it can accommodate the aforementioned nucleic acid molecule.

[0069] Preferably, the expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

[0070] To solve the above technical problem, the fifth aspect of the present application provides a transformant comprising the recombinant expression vector according to the fourth aspect of the present application in a host cell.

[0071] The transformant can be prepared by conventional methods in the art, e.g. by transforming the aforementioned recombinant expression vector into a host cell. The host cell can be any conventional host cell in the art as long as it can satisfy the conditions that the aforementioned recombinant expression vector can be stably replicated by itself and the nucleic acid carried by the aforementioned recombinant expression vector can be effectively expressed. Preferably, the host cell comprises a eukaryotic cell and / or a prokaryotic cell, preferably an E. coli cell such as TG1 or BL21 (for expressing single-chain antibody or Fab antibody) and a eukaryotic cell, preferably a HEK293 cell or a CHO cell (for expressing full-length IgG antibody). The aforementioned recombinant expression plasmid can be transformed into a host cell to obtain a preferred recombinant expression transformant according to the present application. The transformation method can be any conventional transformation method in the art, preferably chemical transformation, heat shock or electroporation.

[0072] To solve the above technical problem, the sixth aspect of the present application provides a method for preparing an antibody or antigen-binding fragment thereof targeting Sirpα, which comprises culturing the transformant according to the fifth aspect of the present application and obtaining the antibody or antigen-binding fragment thereof targeting Sirpα from the culture.

[0073] To solve the above technical problems, the seventh aspect of the present application provides a pharmaceutical composition comprising the Sirpα-targeting antibody or antigen-binding fragment thereof according to the first aspect of the present application and / or the bispecific antibody according to the second aspect of the present application, and a pharmaceutically acceptable carrier.

[0074] Preferably, the pharmaceutical composition further comprises another anti-tumor antibody as an active ingredient.

[0075] The pharmaceutically acceptable carrier can be a conventional carrier in the art, which can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical adjuvant is a conventional pharmaceutical adjuvant in the art, preferably including a pharmaceutically acceptable excipient, a filler or a diluent, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned antibody or antigen-binding fragment thereof and / or bispecific antibody, and 0.01-99.99% of the pharmaceutical carrier, the percentage being the mass percentage of the pharmaceutical composition.

[0076] Preferably, the pharmaceutical composition is an anti-tumor drug. For example, a drug for treating breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome, etc. blood tumors and solid tumors.

[0077] The administration route of the pharmaceutical composition of the present application is preferably injection administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc. The pharmaceutical composition is various dosage forms in the art, preferably in the form of solid, semi-solid or liquid, i.e. aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally or intramuscularly. Preferably, the pharmaceutical composition can also be administered as an aerosol or coarse spray, i.e. nasally; or intrathecally, intramedullary or intraventricularly. More preferably, the pharmaceutical composition can also be transdermally, percutaneously, topically, enterally, intravaginally, sublingually or rectally administered.

[0078] The dosage levels of the pharmaceutical compositions described herein can be adjusted according to the amount of the composition required to achieve the desired diagnostic or therapeutic result. The administration regimen can also be a single injection or multiple injections, or adjusted. The dosage level and regimen selected depend on a variety of factors, including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health and prior medical history of the subject to be treated, among other factors.

[0079] For combination therapy, the above-mentioned antibody or antigen-binding fragment thereof targeting Sirpa, the above-mentioned bispecific antibody and / or the additional therapeutic or diagnostic agent can each be used as a single agent, within any time frame appropriate to perform the intended treatment or diagnosis. Thus, these single agents can be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours of each other) or in sequential succession. For example, these single agents can be administered within a year, or 10, 8, 6, 4, or 2 months, or 4, 3, 2, or 1 week, or 5, 4, 3, 2, or 1 day.

[0080] For additional guidance regarding formulations, dosages, administration regimens, and measurable outcomes of treatment, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, among other works.

[0081] To solve the above technical problems, the eighth aspect of the present application provides a use of the antibody or antigen-binding fragment thereof targeting Sirpa according to the first aspect of the present application, the bispecific antibody according to the second aspect of the present application, or the pharmaceutical composition according to the seventh aspect of the present application in the preparation of a drug for diagnosing, preventing and / or treating a tumor. Preferably, the tumor is as described in the seventh aspect of the present application.

[0082] To solve the above technical problems, the eighth aspect of the present application provides a use of the antibody or antigen-binding fragment thereof targeting Sirpa according to the first aspect of the present application, the bispecific antibody according to the second aspect of the present application, or the pharmaceutical composition according to the seventh aspect of the present application in the preparation of a drug for diagnosing, preventing and / or treating a tumor. Preferably, the tumor is as described in the seventh aspect of the present application.

[0083] To solve the above technical problems, the present application also provides a kit-of-parts, which comprises a kit A and a kit B, the kit A is the Sirpα-targeting antibody or antigen-binding fragment thereof according to the first aspect of the present application, the bispecific antibody according to the second aspect of the present application or the pharmaceutical composition according to the seventh aspect of the present application, and the kit B is another anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody. The kit A and the kit B can be used simultaneously, or the kit A can be used first and then the kit B, or the kit B can be used first and then the kit A, which can be determined according to the actual needs of specific applications.

[0084] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. And, the cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory operation steps used herein are the conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0085] In the present application, the term "variable" generally refers to the fact that certain portions of the variable domain sequence of an antibody vary strongly, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments of the light chain and the heavy chain variable region, which are called complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called framework regions (FR). The variable domains of the natural heavy and light chains each comprise four FRs, mostly adopting a β-sheet conformation, connected by three CDRs, forming loops connecting, and in some cases forming part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FRs, and together with the CDRs from the other chain form the antigen binding site of the antibody, and the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example, participating in antibody-dependent cellular cytotoxicity.

[0086] The three-letter code and one-letter code of amino acids used in the present application are well known to those skilled in the art, or as described in J. Biol. Chem, 243, p3558 (1968).

[0087] As used herein, the term "comprising" or "including" is intended to mean that the compositions and methods include the recited elements, but do not preclude other elements, but according to the understanding of the context, it also includes the case of "consisting of".

[0088] The term "epitope" refers to the portion of an antigen (e.g., Sirpa) that specifically interacts with an antibody molecule. The term "compete" refers to the ability of an antibody molecule to interfere with the binding of an anti-Sirpa antibody molecule to a target (e.g., Sirpa). The interference with binding can be direct or indirect (e.g., through allosteric modulation of the antibody molecule or the target). The extent to which an antibody molecule is able to interfere with the binding of another antibody molecule to its target can be determined using a competitive binding assay (e.g., a FACS assay, an ELISA, or a BIACORE assay).

[0089] The term "antibody" as used herein includes immunoglobulins (Ig) which are four polypeptide chains of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The immunoglobulin heavy chain constant region has different amino acid compositions and arrangement sequences, and thus has different antigenicities. Accordingly, the immunoglobulin can be classified into five types, or called isotypes, i.e., IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same type of Ig can be further classified into different subtypes according to the differences in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, such as IgG1, IgG2, IgG3 and IgG4. The light chain is classified into κ chain or λ chain by the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0090] The sequences of about 110 amino acids at the N-terminus of the heavy and light chains are highly variable and form the variable region (V region); the remaining amino acid sequences are relatively stable and form the constant region (C region). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDRs and 4 FRs, and the order of arrangement from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are VL CDR1, VL CDR2 and VL CDR3; the 3 CDRs of the heavy chain are VH CDR1, VH CDR2 and VH CDR3.

[0091] Within the light chain and the heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain further contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of VH and heavy chain constant region (abbreviated as HC or CH). The heavy chain constant region is composed of 3 domains (abbreviated as HC1, HC2 and HC3 or CH1, CH2 and CH3, respectively). Each light chain is composed of VL and light chain constant region (abbreviated as LC or CL). Generally, one light chain includes one light chain constant region. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0092] The term "murine antibody" in the present application is a monoclonal antibody against human Sirpα prepared according to the knowledge and skill in the art. The subject is injected with Sirpα antigen to prepare, and then hybridomas expressing antibodies having the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present application, the murine Sirpα antibody or antigen-binding fragment thereof can further comprise a light chain constant region of murine κ, λ chain or variant thereof, or further comprise a heavy chain constant region of murine IgGl, IgG2, IgG3 or IgG4 or variant thereof.

[0093] The term "chimeric antibody" is an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, a hybridoma secreting a murine monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the resulting human antibody is cloned as needed. The mouse variable region gene is linked to the human constant region gene to form a chimeric gene, which is inserted into a vector, and finally the chimeric antibody molecule is expressed in a eukaryotic cell, an industrial system or a prokaryotic industrial system. In a preferred embodiment of the present application, the antibody light chain variable region of the Sirpα chimeric antibody further comprises a light chain FR of murine κ, λ type or variant thereof. The antibody heavy chain variable region of the Sirpα chimeric antibody further comprises a heavy chain FR of murine IgGl, IgG2, IgG3, IgG4 or variant thereof. The constant region of the human antibody can be selected from the heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or variant thereof, preferably comprising the heavy chain constant region of human IgGl or IgG4, or IgGl with altered ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement dependent cytotoxicity) activity after amino acid mutation. The ADCC and CDC effector functions of the antibody can be reduced or eliminated, or enhanced, by modification of the Fc fragment of IgG. The modification refers to mutation in the heavy chain constant region of the antibody, such as N297A, L234A or L235A selected from IgGl; IgG2 / 4 chimera, F235E, L234A / E235A, F243L, or S239D / A330L / I332E mutations of IgG4, etc.

[0094] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody in which the CDR sequences of a mouse are grafted into a human antibody variable region framework. In particular, the CDRs of the Sirpa antibodies of the present application, defined as each CDR sequence according to the CCG, Kabat, AbM, Chothia or Contact numbering scheme, are grafted into a human antibody variable region framework. Preferably, the light chain and / or the heavy chain of the humanized Sirpa antibodies of the present application can comprise 0-10 back mutations. Human antibody germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) www.imgt.org and www.vbase2.org.

[0095] As used herein, the term "specifically binds" with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species can also bind the antigen from one or more species. However, such cross-reactivity between species does not alter by itself the classification of the antibody according to the specificity. In another example, an antibody that specifically binds an antigen can also bind different allelic forms of the antigen. However, such cross-reactivity does not alter by itself the classification of the antibody according to the specificity. In some instances, the term "specifically binds" or "binds specifically" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species.

[0096] The term "deamidation" refers to the removal of an amino group at a site or at a certain site of a molecule. "Deamidation sensitive site" refers to a molecule and a certain site of a molecule that is more susceptible, more prone to deamidation.

[0097] The term "antigen binding fragment" refers to antigen binding fragments and antibody analogs of antibodies that generally include at least a portion of an antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. Antibody fragments retain at least certain binding specificities of the parental antibody. Generally, antibody fragments retain at least 10% of the binding activity of the parent on a molar basis. Preferably, antibody fragments retain at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0098] Antibody molecules include diabodies and single chain molecules as well as antigen binding fragments of antibodies (e.g., Fab, F(ab')2, scFv, and Fv). Antibody molecules include or consist of one heavy chain and one light chain (referred to herein as half-antibodies). Fab', F(ab')2, Fc, Fd, Fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their corresponding antigen or receptor. Antibodies and antibody fragments can be from any antibody class, including, but not limited to, IgG, IgA, IgM, IgD, and IgE and from any antibody subclass. Antibody molecules can be monoclonal or polyclonal. Antibodies can also be human antibodies, humanized antibodies, CDR-grafted antibodies, or in vitro generated antibodies.

[0099] A "Fab fragment" consists of one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. An "Fc" region contains the two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. A "Fab' fragment" contains one light chain and a portion of one heavy chain that includes the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains that include the constant region between the CH1 and CH2 domains, such that interchain disulfide bonds form between the two heavy chains. Thus, an F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the heavy chains. The term "Fv" is intended to mean the antibody fragment consisting of VL and VH domains of a single arm of an antibody, but lacking the constant regions.

[0100] In some cases, an antigen-binding fragment of an antibody is a single chain binding fragment (e.g., scFv), in which the VL and VH domains are paired by a linker that enables them to be produced as a single polypeptide chain [see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating G4S amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (G4S)4or (G4S)3may be used, but variants thereof can also be used.

[0101] The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; antibodies having two or more VL and VH regions, each VH-VL unit binds to a different target or to a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binds to a different target or to a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, and triabodies, antibody fragments covalently or noncovalently linked together, and the like.

[0102] The antibodies disclosed herein can also be single domain antibodies. Single domain antibodies can include antibodies whose complementarity determining regions are part of a single polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be any of the art, or any future single domain antibody. Single domain antibodies can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. According to some aspects, the single domain antibody is a naturally occurring single domain antibody, known as a heavy chain antibody lacking a light chain. For clarity reasons, such variable domain derived from a heavy chain antibody naturally lacking a light chain is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in Camelidae species (e.g., camel, llama, dromedary, alpaca, and guanaco). Other species than camel can produce heavy chain antibodies naturally lacking a light chain, and such VHH are contemplated.

[0103] Monoclonal antibodies or antigen binding fragments can be recombinantly produced using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or other art-known techniques. Methods for producing and purifying antibodies and antigen binding fragments are well known in the art and can be found in, for example, Current Protocols in Protein Science. Antigen binding fragments can also be prepared using conventional methods.

[0104] "Identity," "homology," "variant sequence," "mutation" refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions in the comparison, times 100. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. In general, the comparison is made at the level of the nucleotide or amino acid sequence. "Optimization" refers to mutations that preserve or improve the binding of the antibody to the antigen, in the present case, mutations that preserve, maintain or improve binding to Sirpα.

[0105] The terms "polypeptide," "peptide," and "protein" (if single chain) are used interchangeably herein. The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably.

[0106] The term "mutation" includes substitution, insertion and / or deletion of an amino acid or a nucleotide, "amino acid substitution" and "conservative amino acid substitution" are respectively where an amino acid residue is replaced with another amino acid residue and with an amino acid residue having a similar side chain.

[0107] "Lentivirus" as used herein refers to the genus Lentivirus under the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of a host cell, thus they are one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses. Vectors from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0108] The term "recombinant expression vector" as used herein is a composition comprising an isolated nucleic acid and is useful for delivering the isolated nucleic acid into the interior of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0109] The expression "cell", "cell line" used in the present invention are used interchangeably and all such designations include the progeny. The term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as E. coli, fungal cells such as yeast cells, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0110] The term "transfection" refers to the introduction of foreign nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0111] "Optional", "any", "any one", "any" or "any" means that the event or environment described subsequently can but does not necessarily occur, the description including the event or environment occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that the antibody heavy chain variable region of a particular sequence can but does not necessarily exist. "One" and "one" are used in the present invention to refer to one or more than one grammatical object. Unless the context clearly indicates, the term "or" is used in the present invention to mean the term "and / or" and is used interchangeably therewith. "About" and "approximately" shall generally mean an acceptable degree of error, given the nature or precision of the measurement. An exemplary degree of error is within 10% of the stated value and more generally within 5% of the stated value. The methods and compositions disclosed herein encompass polypeptides and nucleic acids having the specified sequence, variant sequences or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95%, 99% or more identical to the specified sequence. In the case of amino acid sequences, the term "substantially identical" is used in the present invention to refer to a first amino acid sequence.

[0112] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, i.e., the concentration that elicits 50% of the maximal effect. The term IC 50 refers to the half-inhibitory concentration, i.e., the concentration of inhibitor required to inhibit 50% of the activity of an enzyme, cell, cell receptor, or growth of a microorganism, etc.

[0113] The pharmaceutical composition of the present invention can be prepared into various dosage forms as needed, and the dosage that is beneficial to the patient can be administered by a physician according to the patient's species, age, weight, and general disease condition, administration method, etc. The administration method can be, for example, injection or other treatment.

[0114] The methods, compositions, combination therapies described herein can be combined with other active agents or treatment modalities, the methods comprising administering to a subject an anti-Sirpa antibody molecule described herein, optionally in combination with one or more inhibitors of PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibodies (immunotherapies) or other oncology therapeutic antibodies, Her-2, EGFR, VEGF, VEGFR, CLDN18.2 antibodies, etc., and ADCs (such as T-DM1), bispecific antibodies, chemotherapeutics, etc., in amounts or dosages that are higher than, lower than, or equal to the amounts or dosages of each active agent used alone (e.g., as monotherapy). The anti-Sirpa antibody, additional active agent, or both, can be administered in amounts or dosages that are lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amounts or dosages of each active agent used alone (e.g., as monotherapy).

[0115] The anti-Sirpa antibody molecule can be administered in combination with one or more of the following: immune-based strategies, targeted drugs (e.g., VEGF inhibitors such as monoclonal antibodies to VEGF); VEGF tyrosine kinase inhibitors such as sunitinib, sorafenib, and apatinib; RNAi inhibitors or inhibitors of downstream mediators of VEGF signaling, e.g., inhibitors of mammalian target of rapamycin (mTOR).

[0116] As used herein, the terms "carcinoma", "cancer", "tumor" are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

[0117] The above-mentioned preferred conditions can be combined in any way, to obtain preferred embodiments of the present application. The reagents and starting materials used in the present application are commercially available.

[0118] The positive progress effect of the present application is that:

[0119] 1) Compared with the prior art, the antibody or its antigen-binding fragment of the present invention has good binding activity with Sirpα. It can not only block the binding of human Sirpα (including Sirpα-V1 and Sirpα-V2) to human CD47, making the antibody or its antigen-binding fragment usable as a new drug to target Sirpα and CD47 binding to achieve the purpose of treating tumors, but also the antibody or its antigen-binding fragment of the present invention can simultaneously bind to both forms of human Sirpα, Sirpα-V1 and Sirpα-V2, and the binding activity is significantly higher than that of the prior art. Therefore, the antibody or its antigen-binding fragment of the present invention has many advantages as a drug development tool and can target more patient groups (people expressing Sirpα-V1 and / or people expressing Sirpα-V2).

[0120] 2) The antibodies or antigen-binding fragments of the present invention do not bind to human Sirpβ and Sirpγ; nor do they bind to human T cells. This makes the antibodies or antigen-binding fragments of the present invention more selective, avoiding the side effects caused by non-targeting effects resulting from binding to T cells in clinical practice.

[0121] 3) The antibody or its antigen-binding fragment of the present invention can bind strongly to various polymorphisms of cyno Sirpα, which allows the use of primates (cynomolgus, cyno) in preclinical safety assessment studies, greatly facilitating preclinical pharmacology, toxicology and other research.

[0122] 4) PTM analysis results show that the antibody or its antigen-binding fragment of the present invention has the characteristics of low immunogenicity and low drugability risk.

[0123] 5) The antibodies or antigen-binding fragments of the present invention, after humanization, exhibit higher expression levels, facilitating downstream production and processes and saving costs. In a preferred embodiment of the present invention, the expression level of the humanized antibody reaches as high as 275 mg / L, which is approximately 4.5 times higher than that of its chimeric antibody and the prior art antibody OSE-172.

[0124] 6) The sequence-specific bispecific antibody SBody designed and screened based on the Sirpα antibody sequence of this invention can retain the functional activity of antibodies targeting both targets. Its binding activity to both targets is close to that of its corresponding monoclonal antibody, and its activity in blocking the binding of antigens to their corresponding ligands is consistent with the changes in the binding activity of the corresponding monoclonal antibody. These bispecific antibodies (referred to as SBody in this invention) have structures similar to conventional IgG, possessing the same complete Fc as normal antibodies, allowing their purification process to be carried out as with normal antibodies. Therefore, the process is simple and has the advantage of low production cost.

[0125] In summary, the unique properties of the antibody or antigen-binding fragment thereof of the present application make it more suitable for the development of drugs for antibodies or antigen-binding fragments thereof against human Sirpα target, and can be administered alone or in combination as a candidate drug, especially providing a new and even better choice for combined immunotherapy of tumors such as PD-1 antibody. BRIEF DESCRIPTION OF DRAWINGS

[0126] Figure 1 Figure 1 is the binding activity of the anti-human Sirpα human-mouse chimeric antibody mab14c of the present application and human T cells, in which 1a, 1b, 1c, 1d are the results of negative control, control antibody 1, control antibody 2 and the antibody of the present application, respectively.

[0127] Figure 2 Figure 2 is the specific binding activity of the anti-human Sirpα humanized antibody of the present application and human Sirpβ (left panel) and Sirpγ (right panel) protein.

[0128] Figure 3 Figure 3 is the binding activity of the anti-human Sirpα humanized antibody of the present application and cyno Sirpα (different polymorphism). DETAILED DESCRIPTION

[0129] The present application will be further described by way of examples, but the present application is not limited to the scope of the examples described. The experimental methods in the following examples, if no specific conditions are specified, are selected according to conventional methods and conditions, or according to the instructions of the product.

[0130] Example 1 Cloning, expression and purification of antigen, antibody

[0131] The antigen used in the present application is purchased from different companies as follows.

[0132] Beijing Yiqioshenzhou Technology Co., Ltd.: human Sirpα-V1-his (item number: 11612-H08H), human Sirpα-V1-mFc (item number: 11612-H38H), mouse Sirpα-his (item number: 50956-M08H), human Sirpγ (item number: 11828-H08H); or Beijing Baipusaisi Biotechnology Co., Ltd.: human Sirpβ-hFc (item number: SIA-H5257), human Sirpγ-hFc (item number: SIG-H5253) or Baiying Biotechnology human CD47-his (item number: B2048) or obtained by expression and purification of the present application.

[0133] The sequence of the expressed human Sirpα-V1 protein (his, or Fc Tag) is NCBI Reference Sequence: NP_001035111, with a full length of 504 amino acids, of which the first 30 are signal peptides; the extracellular region (ECD) is amino acids 31-373. Among them, amino acids 31-137 of the ECD are the Ig-like-V-type region, amino acids 148-247 are the Ig-like C1-type1 region, and amino acids 254-348 are the Ig-like C1-type2 region.

[0134] The sequence of the human Sirpα-V2 (his, or Fc tag) protein is NCBI Reference Sequence: NP_001317657.1, with a full length of 508 amino acids, of which the first 30 are signal peptides; the ECD is amino acids 31-373. In the extracellular region, amino acids 35-145 are the Ig-like 1 region, amino acids 147-250 are the Ig-like 2 region, and amino acids 252-333 are the Ig-like 3 region.

[0135] The sequence of the human Sirpβ (his tag) protein is NCBI Reference Sequence: NP_006056.2, with a full length of 398 amino acids, of which the first 29 are signal peptides; the ECD region is amino acids 30-371. In the ECD, amino acids 37-144 are the Ig-like-V-type region, amino acids 142-253 are the Ig-like region, and amino acids 265-344 are the Ig C region.

[0136] The sequence of the human Sirpγ (his tag) protein is NCBI Reference Sequence: AAH64532.1, with a full length of 387 amino acids, of which the first 28 are signal peptides; the ECD is amino acids 29-360. In the ECD, amino acids 29-137 are the Ig-like-V-type region, amino acids 146-245 are the Ig-like C1-type1 region, and amino acids 252-340 are the Ig-like C1-type2 region.

[0137] The sequence of the NOD mouse Sirpα (his tag) protein is referenced in the article "polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells" (nature immunology, 2007).

[0138] Cyno Sirpα (cynomolgus Sirpα, Macaca Sirpα) has different polymorphism forms. In addition to two sequences from NCBI public database, four polymorphism sequences of Cyno Sirpα different from NCBI public database were obtained by collecting different cynomolgus monkeys. The binding activity of the antibodies of the present application and the different polymorphism forms of Cyno Sirpα protein was evaluated.

[0139] Cyno Sirpα (his tag) protein sequence from NCBI database (see https: / / www.ncbi.nlm.nih.gov / ), Reference Sequence: NP_001271679.1, full length of 503 amino acids, of which the first 30 amino acids are signal peptide; the ECD is the 31st-370th amino acid. In the ECD, the 38th-144th amino acid is the Ig-like-V-type region, the 142nd-253rd amino acid is the Ig region, and the 265th-345th amino acid is the Ig-C region. NCBI Reference Sequence: XP_015313155.1, full length of 503 amino acids. The ECD sequences of the two are different by 3 amino acids (polymorphism).

[0140] In addition, the present application purchases cynomolgus monkey (cyno) blood samples from Zhaoqing Experimental Animal Center, separates human peripheral blood mononuclear cells (PBMC) cells by using SEPMATE 50 (Beijing Branch of Canadian Stem Cell Technology Co., Ltd., Item No. 86450), and then cultures the PBMC cells in RPMI1640 (HYCLONE, Cat. No. SH30809.01) containing 10% FBS (Shanghai Bossing Biological Technology Co., Ltd., Cat. No. BS-0002-500), adheres for 3 hours, removes the suspended lymphocytes in the supernatant, digests the adherent macrophages by using trypsin (Shanghai Yuancheng Biological Technology Co., Ltd., Cat. No. S310JV), extracts RNA by using the trizol method, reversely transcribes the target fragment after PCR amplification, uses the primers SI-2F: taaacggatctctaGCGAATTCatggagcccgccggcccggcccccg (SEQ ID NO: 1) and SI-2R: cggccttgccggcctcGAGCGGCCGCtgtctgattcggacgaggtagag (SEQ ID NO: 2) for PCR, clones the purified PCR product to a vector for sequencing, and uses the sequencing primer p63a-SEQ: cacaggtgtccactcccaggt (SEQ ID NO: 49). Finally, two different cyno Sirpα sequences (polymorphism) different from the above-mentioned NCBI disclosed cyno Sirpα sequence are obtained. At the same time, two different cyno Sirpα sequences (polymorphism) are obtained from the cynomolgus monkey blood samples purchased from Hainan Animal Center by the same method.

[0141] The base sequences of the full gene synthesis (C-terminal connection 6×his) of each cyno Sirpα protein are cloned into the pTT5 vector (Biovector, Cat#: 102762) for expression and purification to obtain different cyno Sirpα proteins.

[0142] Table 1 Different polymorphic molecules of cyno Sirpα

[0143] Cyno Sirp alpha protein number Sequence number L932 NP_001271679.1 L933 XP_015313155.1 L936 RB3-3 L937 RB3-5 L938 RB6-1 L939 RB6-2

[0144] The amino acid sequences of the cyno Sirpα proteins (polymorphism) found by the present application are as follows:

[0145] RB3-3 sequence:

[0146] EEELQVIQPEKSVSVAAGDSATLNCTVTSLIPVGPIQWFRGAGPGRELIYHQKEGHFPRVTSVSESTKRNNMDFSIHISNITPADAGTYYCVKFRKGSPDVEVKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVICEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVTKFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRDDVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN (SEQ ID NO: 3):

[0147] RB3-5 sequence:

[0148] EEELQVIQPEKSVSVAAGDSATLNCTVSSLIPVGPIQWFRGAGPGRELIYNLKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPRDITLKWFKNGNELSDVQTNVDPAGKSVSYSIRSTARVLLTRRDVHSQVICEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVTKFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRDDVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN (SEQ ID NO: 4):

[0149] RB6-1 sequence:

[0150] EEELQVIQPEKSVSVAAGESATLNCTATSLIPVGPIQWFRGVGPGRELIYHQKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVICEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVMKFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRDDVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN (SEQ ID NO: 5)

[0151] RB6-2 sequence:

[0152] EEELQVIQPEKSVSVAAGESATLNCTATSLIPVGPIQWFRGVGPGRELIYSQKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVICEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVTKFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRDDVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN (SEQ ID NO: 6)

[0153] The sequence of human CD-47 (hFc / his tag) protein is NCBI Reference Sequence: NP_001768.1, which has a full length of 323 amino acids, wherein the first to 18th amino acids are signal peptide; the ECD is the 19th to 141st amino acids.

[0154] The sequence of human PD-1 (hFc / his tag) protein is NCBI Reference Sequence: NP_005009.2, with a full length of 288 amino acids, wherein the 1st-20th is a signal peptide; and the ECD is the 21st-167th amino acid.

[0155] The sequence of human PD-L1 (hFc / his tag) protein is NCBI Reference Sequence: NP_054862.1, with a full length of 290 amino acids, wherein the 1st-18th is a signal peptide; and the ECD is the 19th-239th amino acid.

[0156] The hFc tag used in the present application is connected with IgG1 Fc region at the C-terminus, and the his tag is connected with 6×his at the C-terminus.

[0157] The antibodies used in the present application, including positive control antibody 1 (referred to as Ref1, OSE-172, the sequence is from WO2017178653A2, #55 light chain, #42 heavy chain, # represents the sequence number in the reference) and positive control antibody 2 (referred to as Ref2, the sequence is from SIRP29 in US20140242095A1, #6 light chain, #12 heavy chain, as a positive control when combined with Sirpγ), are all expressed and purified by the present application.

[0158] The pTT5 vector (Biovector, Cat#: 102762) is used as an expression vector. The expressed recombinant protein, antibody light and heavy chain sequences, are cloned into the pTT5 vector, expressed by transient transfection of HEK293F cells (Life Technologies Cat.No.11625019, hereinafter referred to as 293F cells), and then purified.

[0159] Specifically, the 293F cells are cultured in Gibco FreeStyle 293Expression Medium (Gibco, Cat#: 12338018) medium. Before the start of transfection, the cell concentration is adjusted to 8×10 5 cell / ml, 1%FBS (Aus GeneX FBS Excellent supplier: AusGeneX, China, Cat#: FBSSA500-S), 37℃ 8%CO2 shaker culture for 24h, and the survival rate is >95% by microscopic examination again, and the cell concentration is 1.2×10 6 cell / ml.

[0160] Preparation of 300 ml culture system cells, 15 ml Opti-MEM (Gibco, Cat#: 31985070) is dissolved in 150 μg of heavy chain, light chain plasmid or fusion protein plasmid (if it is a recombinant protein, the amount of single plasmid is 300 μg), 0.22 μm filter sterilization. Then take 15 ml Opti-MEM and dissolve 1 mg / ml PEI (Polysciences, Inc, Cat#: 23966-2) 600 μl, and then stand for 5 min. Slowly add PEI to the plasmid, incubate at room temperature for 10 min, slowly drop the plasmid PEI mixed solution into the culture bottle while shaking, and culture at 37℃ with 8% CO2 for 5 days to collect the sample, 3300G 10 min to collect the supernatant for purification.

[0161] Antibody or -Fc fusion protein purification: Centrifuge the sample at high speed to remove impurities, and equilibrate the gravity column (Shenguo Biotechnology, Cat#: F506606-0001) containing Protein A (Mabselect, GE Healthcare Life Science, Cat#: 71-5020-91AE) with PBS pH 7.4, and flush with 2-5 times the column volume. Pass the sample through the column. Flush the column with 5-10 times the column volume of PBS (Shenguo Biotechnology, Cat#: B548117-0500). Then elute the target protein with 0.1M acetic acid at pH 3.5, and then adjust to neutral with Tris-HCl at pH 8.0. Determine the concentration by enzyme label meter, and store for future use.

[0162] His Tagged protein purification: Centrifuge the sample at high speed to remove impurities. Equilibrate the nickel column (Ni smart beads 6FF Changzhou Tiandi Renhe Biotechnology Co., Ltd., Cat#: SA036010): equilibrate the nickel column with PBS pH 7.4 containing 10 mM imidazole and 0.5M NaCl, and flush with 2-5 times the column volume. Pass the sample supernatant through the column. Flush the impurities: use PBS pH 7.4 containing 10 mM imidazole and 0.5M NaCl to flush the column, remove the non-specifically bound impurities, and collect the effluent. Elute the target protein with PBS pH 7.4 containing 250 mM imidazole and 0.5M NaCl. Buffer exchange: centrifuge the eluted target protein through an ultrafiltration tube at 12000g for 10 min (ultrafiltration tube, Merck Millipore, Cat#: UFC500308), and then add 1 ml of PBS. Determine the concentration, and store for future use.

[0163] Example 2 Construction of high expression cell lines and cell activity (ELISA) detection

[0164] The high expression cell strains used in the present application are all constructed by the present inventors through the stable cell strain construction platform of the company. The construction process is described below by taking the construction of human Sirpα high expression cell strain as an example. The specific steps are as follows:

[0165] On the first day of the experiment, 293T cells (China Institute of Cell Culture Preservation Committee, Cat#: GNHu17) were inoculated in two 6cm culture dishes, and the number of cells in each culture dish reached 7.5x10 5 On the second day, 4μg of plasmid (pGag-pol, pVSV-G, etc. BioVector plasmid vector strain cell gene preservation center) and plasmid pBabe-hSirpα containing human Sirpα gene were added to 200μl of OPTI-MEM (Thermofisher Scientific, Cat#: 31985070), and 36μl of transfection reagent fectin (Shanghai Yuenpui Biotechnology Co., Ltd., Cat#: F210) was prepared in 200μl of OPTI-MEM. The two were mixed and incubated at room temperature for 5min, then the mixture (200μl per dish) was added to the cultured 293T cells. On the third day, the 293T cell culture solution was replaced with 4ml of DMEM high glucose medium (Shanghai Yuenpui Biotechnology Co., Ltd., Cat#: L130KJ). On the fourth day, CHO-K1 cells (China Institute of Cell Culture Preservation Committee, Cat#: SCSP-507) were inoculated in 10cm culture dishes, and the number of cells reached 5x10 5 On the fifth day, the 293T cell supernatant (virus) was collected and filtered through a 0.45μm filter to the cultured CHO-K1 cells, and 10μg / ml polybrene (Shanghai Yewen Biological Technology Co., Ltd., Cat#: 40804ES76) was added. After mixing, it was placed in the incubator for 3-4h, and then replaced with DMEM / F12 10%FBS medium (Shanghai Yuenpui Biotechnology Co., Ltd., Cat#: L310KJ). On the seventh day, the CHO-K1 cells were passaged, and on the eighth day, the passaged cells were added with 10μg / ml puromycin for screening (Shanghai Yuenpui Biotechnology Co., Ltd., Cat#: S250J0). 2-3 days later, the cells died in large numbers, the medium was replaced and the cells were cultured until the cells no longer died, the cells were expanded, and the single clone cell strain was screened, expanded, and cryopreserved.

[0166] The amino acid sequence of human Sirpα (pBabe-hSirpα-V1) used in this example is NP_001035111, full length 1-504 amino acids, wherein 1-30 is the signal peptide; 31-504 is the protein sequence expressed by the CHO-K1 hSirpα+ cell line constructed by the application.

[0167] hSirpα+ cell binding activity (ELISA) detection:

[0168] After the above-mentioned cells, i.e. the human Sirpα high-expression monoclonal cell strain, were expanded and cultured, 10×10 4 / well were plated in a 96-well plate, and after overnight incubation in a 37°C incubator, the supernatant was removed, 100 μl / well of immunostaining fixing solution (Shanghai Biyun Tian Biotechnology Co., Ltd., Cat#: P0098) was added, and the plate was fixed at room temperature for half an hour. After washing once with PBS (Shanghai Yuancheng Biotechnology Co., Ltd., Cat#: B320), 230 μl of 5% milk was added for blocking at 37°C for 2 hours, and the plate was washed 3 times with PBST. 50 μl of the sample to be tested was added to each well, and the sample was diluted 5 times. After incubation at 37°C for 1 hour, the plate was washed 5 times with PBST. 50 μl of Anti-human HRP (Jackson Immuno Research, Cat#: 109-035-003) diluted 1:2500 was added to each well, and the plate was incubated at 37°C for 1 hour. After washing 5 times with PBST, 50 μl of TMB (Surmodic, Cat#: TTMB-1000-01) was added to each well for color development, and 50 μl / well of 1M H2SO4 was added to stop the reaction. The plate was read on an enzyme-labeled instrument (Multiskan GO Thermo, model 51119200), and the data were analyzed using Graphpad prism 5.

[0169] Example 3 Anti-Sirpα antibody and antigen binding experiment (ELISA)

[0170] Different antigens (recombinant proteins) such as human Sirpα-V1-hFc, Sirpα-V1-his, Sirpα-V2-his, Sirpβ-his, Sirpγ-his, monkey Sirpα-his (cyno Sirpα-his) or NOD-mSirpα-his described in Example 1 were diluted with PBS buffer at pH 7.4 to a concentration of 1 μg / ml, 2 μg / ml or 5 μg / ml, and added to a 96-well enzyme plate (Corning, CLS3590-100EA) at a volume of 50 μl / well, and incubated in a 37°C incubator for 2 hours. After discarding the liquid, 230 μl / well of 5% skim milk (Guangming skim milk powder) blocking solution diluted with PBS was added, and incubated in a 37°C incubator for 3 hours or at 4°C overnight (16-18 hours) for blocking. After discarding the blocking solution and washing the plate 5 times with PBST buffer (PBS containing 0.05% tween-20 at pH 7.4), 50 μl / well of supernatant (containing test antibodies) or 10 μg / ml of the test antibody diluted 5-fold in gradient was added, and incubated at 37°C for 1 hour, washed the plate 5 times with PBST, and added 50 μl / well of 1:2500 diluted Anti-mouse or human HRP secondary antibody (Jackson Immuno Research, Cat#: 115-035-003 or 109-035-003) and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well of TMB color developing substrate (KPL, 52-00-03) was added, and incubated at room temperature for 10-15 min, 50 μl / well of 1M H2SO4 was added to stop the reaction, and the absorbance value was read at 450 nm using a MULTISKAN Go microplate reader (ThermoFisher, 51119200). The clones with high binding activity were selected according to the OD value or the EC 50 value was calculated.

[0171] Example 4 Anti-Sirpα antibody prevents Sirpα and CD47 binding activity experiment

[0172] Human Sirpα-V1-hFc / Sirpα-V1-his were diluted to 5 μg / ml or 2 μg / ml in PBS buffer pH 7.4, respectively, and added to 96-well enzyme-linked immunosorbent assay (ELISA) plates (Corning, CLS3590-100EA) at a volume of 50 μl / well, and incubated in a 37 °C incubator for 2 hours. After discarding the liquid, 230 μl / well of 5% skimmed milk (Guangming skimmed milk powder) blocking solution diluted in PBS was added, and incubated in a 37 °C incubator for 3 hours or at 4 °C overnight (16-18 hours) for blocking. After discarding the blocking solution, the plates were washed 5 times with PBST buffer (PBS pH 7.4 containing 0.05% tween-20), and 25 μl / well of supernatant (containing detection antibodies) or 100 μg / ml of the antibody to be tested, which was diluted by a factor of 3, and 25 μl / well of biotin-labeled CD47-hFc at a concentration of 4 μg / ml or CD47-his at a concentration of 3 μg / ml were added, mixed, and incubated in a 37 °C incubator for 1 hour. After washing the plates 5 times, 50 μl / well of 1:1000 diluted streptavidin-HRP secondary antibody (genscript, M00091) or anti-his-HRP secondary antibody (genscript, A00612) was added, and incubated in a 37 °C incubator for 1 hour. After washing 5 times with PBST, 50 μl / well of TMB (Surmodic, Cat#: TTMB-1000-01) was added for color development, and 50 μl / well of 1M H2SO4 was added to stop the reaction. The readings were taken using an enzyme-linked immunosorbent assay (ELISA) reader (Multiskan GO Thermo, model 51119200), and the data were analyzed using Graphpad prism 5.

[0173] Example 5 Anti-human Sirpα antibody binding to human T cells experiment

[0174] The T cells used in the present application were isolated from the peripheral blood of healthy volunteers using SEPMATE 50 (Canadian Stem Cell Technologies Beijing Branch, item number 86450), and the frozen stock solution (RPMI1640 medium: FBS: DMSO = 5:4:1) was stored in a liquid nitrogen tank for standby use. The frozen PBMC cells were taken and added to 3 ml of FACS buffer (1x PBS + 2% FBS), centrifuged at 1000 rpm for 5 min, and after removing the supernatant, resuspended in FACS buffer for counting and adjusting the density to 1x10 6cells / ml. PBMC was added into U-shaped 96-well plate at 100 μl cells / well, and the antibody to be tested was added to the cells to a final concentration of 10 μg / ml, 1 μg / ml and 0 μg / ml, respectively. After mixing, incubate at room temperature for 20 min, centrifuge at 2000 rpm for 5 min. Remove the supernatant, resuspend with 100 μl FACS buffer, centrifuge at 2000 rpm for 5 min. Remove the supernatant, add 50 μl of 1:200 diluted PE anti-human IgG Fc (Biolegend, item number: 409304), mix, and incubate at room temperature in the dark for 20 min. Centrifuge at 2000 rpm for 5 min. Remove the supernatant, wash once with 100 μl FACS buffer, resuspend the cells with 100 μl FACS buffer, and read with a flow cytometer (Novo sampler TM pro, model: NS200). The experimental data was processed with NovoExpress, and the sample well with an antibody concentration of 0 μg / ml was used as the negative control. Based on this, the proportion of T cells specifically bound by the antibody to be tested was calculated.

[0175] Example 6 Discovery of anti-human Sirpα antibody

[0176] The present application uses human Sirpα-V1-mFc (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., item number: 11612-H38H) and hSirpα-V2-hFc (as expressed in Example 1) recombinant proteins as antigens, and cross-immunizes mice with Freund's complete adjuvant for 4 times. After electrofusion and screening of hybridoma, clones with good binding activity to hSirpα-V1 were screened from tens of thousands of hybridoma clones. Further screening unexpectedly found that some clones could bind to both human Sirpα-V1 and human Sirpα-V2, had blocking activity, and did not bind to human PBMC (T cells). Further, through multiple subcloning from these unexpectedly discovered hybridoma clones, monoclonal cell strains were obtained, the antibody sequences were extracted, and the mouse-derived antibodies of the present application were obtained by expression and purification.

[0177] Specifically, SJL mice, female, 4 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (animal production license number: SCXK (Jing) 2016-0011). After the mice were purchased, they were raised in the laboratory environment for 1 week, with day light / night dark cycle adjustment, temperature 20-25℃; humidity 40-60%. The mice were divided into 3 / group / cage. The mice were immunized with the antigen prepared in Example 1. The first immunization adjuvant was Freund's complete adjuvant (Sigma-Aldrich, SIGMA F5506-10M), and the adjuvant from the second immunization was Freund's incomplete adjuvant (Sigma-Aldrich, SIGMA F5881-10M). The ratio of antigen to adjuvant was 1:1 (v / v). The first immunization was 100 μl / 25 μg per mouse, and the second, third, and fourth immunizations were 100 μl / 12.5 μg per mouse, intramuscular injection in the leg. Three days before fusion, 100 μl / 25 μg per mouse was used for booster immunization. The immunization time was day 0, 14, 28, 42, and 56 (booster immunization). On day 36, 50, the ELISA method of Example 3 above was used to detect the antibody titer in the serum of the mice, and the mice with high antibody titer and titer in the plateau phase were selected for spleen cell fusion. The spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (ATCC, CRL-8287) to obtain hybridoma cells, which were then screened to obtain preferred clones. CRL-8287 TM ) were fused to obtain hybridoma cells, which were then screened to obtain preferred clones.

[0178] The hybridoma cell lines were subjected to primary screening, and the binding activity of the antibodies secreted in the supernatant of the hybridoma cell lines and human Sirpα was detected by the ELISA method of Example 3 (part of the binding data is shown in Table 2), and the clones with good activity were selected. The supernatant was taken and the binding activity of the secreted antibodies to hSirpα and hCD47 (blocking activity) was detected by the method described in Example 4, and the binding activity to human Sirpα-V2 was also detected. Preferred clones were further subjected to limited dilution to obtain monoclonal antibody cell lines, and part of the results are shown in Table 3.

[0179] Table 2 Binding activity (OD450 value) of hybridoma fusion cell (clone) supernatant to hSirpα-V1

[0180]

[0181]

[0182] Table 3 Binding activity and blocking activity data (OD450 value) of hybridoma fusion cell (clone) supernatant to hSirpα-V2

[0183]

[0184] Part of the primary screening data is listed in Table 2. The data shows that many hybridoma fusion cells exhibit high binding activity in the initial screening, ELISA reading (OD450 is above 2), such as 4C1, 8C4, 8H7, 9H1 and other clones. However, further screening found that almost all clones either have low binding activity with hSirpα-V2 or have no blocking activity of blocking hSirpα and hCD47 binding (the lower the OD450 value and the greater the difference from the ELISA value, the better the blocking activity), see Table 3.

[0185] It is very unexpectedly found that the clone 14H6 (Table 2, OD450 value 1.81) with good hSirpα-V1 binding activity exhibits strong binding activity with hSirpα-V2 (Table 3, OD450 value 1.25) and can well block the binding of hSirpα and hCD47 (OD450 value 0.33).

[0186] The clone was subjected to multiple limiting dilutions, each dilution for 7-10 days, and after the clone proliferation, the binding activity and blocking activity of the antibodies (supernatant) secreted by each clone with different Sirpα were detected by ELISA method, and the results are shown in Table 4.

[0187] Table 4 Preferred hybridoma cell subclone screening activity

[0188]

[0189] The above results show that the antibodies secreted by the monoclonal cell strains obtained by multiple subcloning of 14H6 retain the binding activity with hSirpα-V1 and hSirpα-V2, and exhibit the blocking activity of hSirpα-V1-his and hCD47-Fc (dimer) binding activity and the blocking activity of hSirpα-V1-hFc (dimer) and hCD47-his binding activity.

[0190] More unexpectedly, the antibodies secreted by these monoclonal cell strains do not bind Sirpγ (the hSirpγ binding activity value in Table 4 is below 0.1, which is the background value). It is shown that the antibodies of the application have very good selectivity for Sirpα and Sirpγ.

[0191] The application further extracts the antibody sequence from one of the subclones of 14H6, 14H6E5B5C8G2, to obtain the preferred mouse mab14 antibody sequence of the application, as follows in the examples.

[0192] Example 7 Extraction, analysis and identification of the mouse anti-human Sirpα antibody mab14 antibody sequence of the application

[0193] The process for extracting antibody sequences from the preferred monoclonal cell strain subcloned from the hybridoma 14H6 is a method commonly used by those skilled in the art. Specifically, the monoclonal cell strain is collected, expanded and cultured, and 1 x 10 6 cells are taken and RNA is extracted using Trizol (Invitrogen, 15596-018) (according to the kit instructions). The extracted RNA is reverse transcribed into cDNA, and the reverse transcription kit is purchased from Shengong Biotechnology (Shanghai) Co., Ltd., Cat#: B532435. The cDNA obtained by reverse transcription is used as a template for PCR amplification. The amplified product is sequenced to obtain the base / coding sequence of the light and heavy chain variable regions of the mab14 antibody, respectively (as follows). The primers used are described in the Novagen published manual TB326 Rev. C0308.

[0194] The base sequence of the light chain variable region of the murine monoclonal antibody mab14 obtained from the preferred hybridoma cell strain of the present application (the underlined portion is the coding sequence) is as follows:

[0195] Taatggtgtccctcagctcagttccttggtctcctgttgctctgttttcaaggtaccagatgt gatat ccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcag gacattaggaattatttaaactggtatcagcagaagccagatggaactgttaaactcctgatctacttcacatcaa cattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacct ggaacaagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaag ctggaaatcaaac gggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttctgaacaactctaccccaaagacatcaaggacct (SEQ ID NO: 7)

[0196] The base sequence of the heavy chain variable region of the murine monoclonal antibody mab14 obtained from the preferred hybridoma cell strain of the present application (the underlined portion is the coding sequence) is as follows:

[0197] Tcatgggatggagctgtatcatgttctttttggtagccgcagctacaggtgtccactccc aggtccat ctgcagcagcctggggctgagcttgtgaagcctggggcttcagtgaagttgtcctgcaaggcttctggctacaatt tcaacatctactggataaattgggtgaagcagaggcctggacaaggccttgagtggattggaaatatttatcctag tagtattagtactaactacaatgagaagttcaagacgaaggccacactgactgtagacaaatcctccaacacagtc tacatgcagttcagcagcctgacatctgaggactctgcggtctattattgtgcgcgatcggagggaacttactatg gtggtcgctacgagggggactggtttggttactggggccaagggactctggtcactgtctctgcagccaaaacaac acccccatcagtctatcc actggcccct gggtgtggag atacaactgg ttcctccgtg actctgggat gcctggtcaa gggctactgc cgagtcgaag ttcc (SEQ ID NO: 8)

[0198] The amino acid sequence encoded by the base sequence of the light chain variable region of the mouse monoclonal antibody mab14 obtained by the hybridoma monoclonal cell strain of the present application is as follows:

[0199] The amino acid sequence of the light chain variable region of the mouse monoclonal antibody mab14 obtained by the preferred hybridoma monoclonal cell strain of the present application is as follows:

[0200] DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYFTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 9)

[0201] The amino acid sequence of the heavy chain variable region of the mouse monoclonal antibody mab14 obtained by the preferred hybridoma monoclonal cell strain of the present application is as follows:

[0202] QVHLQQPGAELVKPGASVKLSCKASGYNFNIYWINWVKQRPGQGLEWIGNIYPSSISTNYNEKFKTKATLTVDKSSNTVYMQFSSLTSEDSAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSA (SEQ ID NO: 10)

[0203] The antibody light chain and heavy chain variable region sequences and the constant regions of different types of IgG, such as human IgG1 (hIgG1), human IgG2 (hIgG2), human IgG3 (hIgG3), or human IgG4 (hIgG4), human light chain κ, λ type; mouse mIgG1, mIgG2, or mIgG3, mouse light chain κ, λ type, and the like, are recombinantly expressed and purified to obtain complete human-mouse chimeric antibodies, mouse antibodies. In the present application, the heavy chain constant region is hIgG4, and the light chain κ type is taken as an example, and the human-mouse chimeric antibody mab14c is obtained by the expression and purification method of Example 1. The binding activity of mab14c to hSirpα-V1, hSirpα-V2, and hSirpα+ cells and the blocking activity of hSirpα and CD47 are detected by the methods of Example 2, Example 3, and Example 4, and compared with the control antibody 1, and the results are shown in Table 5.

[0204] Table 5 Activity analysis of the human-mouse chimeric antibody mab14c of the present invention.

[0205]

[0206] The above results demonstrate that the unexpectedly discovered human-mouse chimeric antibody mab14c not only blocks the binding of Sirpα to human CD47, but also exhibits excellent binding activity with hSIRPα-V1, hSIRPα-V2, and hSirpα+ cells. This differs from Ref1, which does not bind to hSIRPα-V2.

[0207] In addition, the binding activity of mab14c to muSirpα and different polymorphisms of cynoSirpα (including L932, L933, L936-L939) in NOD mice was detected using the method in Example 4, and the results are shown in Table 6.

[0208] Table 6. Analysis of the binding activity of the antibody mab14c of this invention with mouse and cyno Sirpα (ECG) 50 ,nM)

[0209] Sample name NOD-muSirp alpha L932 L933 L936 L937 L938 L939 mab14c No binding 0.10 0.14 0.099 0.099 No binding No binding Ref1 No binding No binding No binding No binding Weak binding Weak binding Weak binding

[0210] Note: EC 50 When greater than 10 nM and less than 50 nM (10 nM ≤ EC) 50 <50 nM) indicates weak binding, EC 50 Between 2 and 10 nM (i.e., 2 nM ≤ EC) 50 When EC50 is <10 nM, it is a weak binding; when EC50 is greater than 50 nM, it is a weak binding. 50 If the signal value is ≥50nM or no signal value can be detected, it is considered non-binding.

[0211] The above results indicate that the antibody mab14c of this invention binds well to four of the six polymorphic proteins of cyno Sirpα, which is significantly different from Ref1, which does not bind to any of the six cyno Sirpα proteins. In the preclinical research stage of new drug development, it is necessary to select relevant primate species for preclinical studies. If the antibody does not bind to primate proteins, such as cyno (the most commonly used primate), then that primate cannot be selected for preclinical studies, which would cause great inconvenience. Therefore, the antibody of this invention binds to multiple polymorphic proteins of cyno Sirpα, which greatly facilitates preclinical studies.

[0212] Furthermore, because human T cells express Sirpγ, in order to evaluate the binding activity (selectivity) of the antibody of the present invention with human T cells, the binding activity of the mab14c antibody of the present invention with human T cells was detected using the method in Example 5. The results are shown in […]. Figure 1. Wherein, la is negative control, i.e. without antibody, only 0.39% of cells (background level) have fluorescence intensity above 10 3 above (positive cells). Namely, no cells with binding were detected. With 10 μg / ml of control antibody 1 (Ref 1) (1b), fluorescence intensity above 10 3 above cells 0.49% (background level), i.e. the antibody does not bind to T cells. Under the same conditions, the positive cells of control antibody 2 (Ref 2) were detected to be 51.3%, i.e. the antibody binds to T cells (1c). Under the same conditions, the positive cell ratio of the antibody of the present application was detected to be 0.9% (background level, 1d), i.e. the antibody of the present application does not bind to T cells.

[0213] The above results show that the antibody mab14c of the present application is a new antibody different from both Ref 1 and Ref 2. It has better selectivity than Ref 1, and can bind to both V1 and V2 forms of Sirpα, so it can be used for more patient groups (people expressing V1 and people expressing V2) in clinic. It has better cyno Sirpα binding activity than Ref 1, which brings great convenience to preclinical pharmacology and toxicology research. In addition, the antibody of the present application is also different from Ref 2, and has better selectivity than Ref 2: it does not bind to Sirpγ or T cells (expressing Sirpγ), which can be used for drug development to avoid the side effects caused by non-specific targeting of T cells in clinic.

[0214] Example 8 Humanization of the murine antibody of the present application

[0215] In order to avoid the risks of immunogenicity and the like in the process of drug development, the murine antibody mab14 of the present application is designed and screened for humanization, and sequence optimization. The specific process is described as follows.

[0216] There are many different methods for defining CDRs in the art, and these CDR defining methods can be summarized as follows in Table 7.

[0217] Table 7 Summary of different methods for defining CDRs of antibodies in the art

[0218] Loop CCG definition Kabat definition AbM definition Chothia definition Contact definition Light chain CDR1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36 Light chain CDR2 L50-L56 L50-L56 L50-L56 L50-L56 L45-L55 Light chain CDR3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96 Heavy chain CDR1 H26-H35 H31-H35 H26-H35 H26-H32 H30-H35 Heavy chain CDR2 H50-H65 H50-H65 H50-H58 H52-H56 H47-H58 Heavy chain CDR3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101

[0219] *For more information, please refer to the website: http: / / www.bioinf.org.uk / abs / #cdrdef

[0220] Wherein, Laa-Lbb in Table 7 can refer to the amino acid sequence from aa position to bb position in the N-terminal of the light chain of the antibody; Haa-Hbb can refer to the amino acid sequence from aa position to bb position in the N-terminal of the heavy chain of the antibody. For example, L24-L34 can refer to the amino acid sequence from 24th position to 34th position in the N-terminal of the light chain of the antibody according to the CCG coding rule; H26-H32 can refer to the amino acid sequence from 26th position to 35th position in the N-terminal of the heavy chain of the antibody according to the CCG coding rule. It is well known to those skilled in the art that there are sometimes insertions and / or deletions in some positions when encoding CDRs.

[0221] The variable region of the above-mentioned murine anti-human Sirpα antibody mab14 can be defined according to various definitions in Table 7, and the CDR sequences are labeled / annotated as follows.

[0222] Table 8 CDR sequences of the anti-hSirpα antibody mab14 of the present application defined according to the CCG numbering rule

[0223]

[0224]

[0225] Table 9 CDR sequences of the anti-hSirpα antibody of the present application defined according to the Kabat numbering rule

[0226] Antibody mab14 CDRs Light chain CDR1 RASQDIRNYLN (SEQ ID NO: 11) Light chain CDR2 FTSTLHS (SEQ ID NO: 12) Light chain CDR3 QQGNTLPWT (SEQ ID NO: 13) Heavy chain CDR1 IYWIN (SEQ ID NO: 17) Heavy chain CDR2 NIYPSSISTNYNEKFKT (SEQ ID NO: 15) Heavy chain CDR3 SEGTYYGGRYEGDWFGY (SEQ ID NO: 16)

[0227] Table 10 CDR sequences of the antibody of the present application defined according to the AbM numbering rule

[0228] Antibody mab14 CDRs Light chain CDR1 RASQDIRNYLN (SEQ ID NO: 11) Light chain CDR2 FTSTLHS (SEQ ID NO: 12) Light chain CDR3 QQGNTLPWT (SEQ ID NO: 13) Heavy chain CDR1 GYNFNIYWIN (SEQ ID NO: 14) Heavy chain CDR2 NIYPSSIST (SEQ ID NO: 18) Heavy chain CDR3 SEGTYYGGRYEGDWFGY (SEQ ID NO: 16)

[0229] Table 11 CDR sequences of the antibody of the present application defined according to the Chothia numbering rule

[0230] Antibody mab14 CDRs Light chain CDR1 RASQDIRNYLN (SEQ ID NO: 11) Light chain CDR2 FTSTLHS (SEQ ID NO: 12) Light chain CDR3 QQGNTLPWT (SEQ ID NO: 13) Heavy chain CDR1 GYNFNIY (SEQ ID NO: 19) Heavy chain CDR2 YPSSI (SEQ ID NO: 20) Heavy chain CDR3 SEGTYYGGRYEGDWFGY (SEQ ID NO: 16)

[0231] Table 12 CDR sequences of the antibody of the present application defined according to the Contact numbering rule

[0232] Antibody mab14 CDRs Light chain CDR1 RNYLNWY (SEQ ID NO: 21) Light chain CDR2 KLLIYFTSTLH (SEQ ID NO: 22) Light chain CDR3 QQGNTLPW (SEQ ID NO: 23) Heavy chain CDR1 NIYWIN (SEQ ID NO: 24) Heavy chain CDR2 WIGNIYPSSIST (SEQ ID NO: 25) Heavy chain CDR3 ARSEGTYYGGRYEGDWFG (SEQ ID NO: 26)

[0233] After the above analysis, labeling and definition of the CDR sequences of the murine antibody mab14 of the present application, humanization is carried out according to the methods disclosed in many literatures in the art. The murine antibody sequence is compared with the human antibody germline database (v-base) to find the human antibody light and heavy chain germlines with high homology, and on this basis, computer modeling is carried out to simulate the positions in the antibody structure that may affect antigen binding, back mutation of key positions and combination, and screening of humanized antibody molecules with preferred activity.

[0234] Specifically, through sequence homology comparison analysis, it is found that the human antibody germline with good homology comparison with the light chain of mab14 includes IGKV1-27*01, IGKV1-33*01, IGKV1-39*01, IGKV1-NL1*01, IGKV1 / OR10-1*01, IGKV1D-33*01, IGKV1D-39*01, IGKV1-12*01, IGKV1-12*02, IGKV1-17*02, etc. Further comparison and analysis, the human antibody germline light chain IGKV1-39*01 is preferred. Sequence alignment finds that the J gene region of the light chain of mab14 has high homology with human antibody germline hJk1, hJk2.1, hJk2.2, hJk2.3, hJk2.4, hJk3, hJk4.1, hJk4.2, hJk5, and further comparison and analysis, hJk4.1 is preferred for the human antibody germline J region of the light chain of mab14 for humanization design, screening and sequence optimization.

[0235] Through sequence homology comparison analysis, it is found that the human antibody germline with good homology comparison with the heavy chain of mab14 includes IGHV1-46*01, IGHV1-46*02, IGHV1-46*03, IGHV1-69*02, IGHV1-69*04, IGHV1-69*06, IGHV1-69*08, IGHV1-69*09, IGHV1-69*10, IGHV1-69*14, etc. Further comparison and analysis, the human germline heavy chain IGHV1-46*01 sequence is preferred for the humanization of the antibody of the application. Sequence alignment finds that the J gene region of the heavy chain of mab14 has high homology with the human antibody germline heavy chain J gene hJh1, hJh2, hJh3.1, hJh3.2, hJh4.1, hJh4.2, hJh4.3, hJh5.1, hJh5.2, hJh6.1, hJh6.2, hJh6.3, etc. Further comparison and analysis, hJh4.1 is preferred for the human antibody germline J region of the heavy chain of the mouse-derived antibody mab14 of the application for humanization design, screening and sequence optimization.

[0236] The CDRs of the antibody of the present application are grafted to the selected humanized light and heavy chain human antibody germline templates of mab 14 (see the definition of CDRs above, mainly according to the CCG definition in this embodiment) and then recombined with the IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the mouse antibody, back-mutations are performed on the buried residues, residues directly interacting with the CDRs, and residues having a significant influence on the conformation of VL and VH, and the effects of these mutations and combinations of mutations on the activity of the antibody are screened, and the CDRs are optimized for chemically unstable amino acid residues, to obtain an antibody molecule sequence that is optimized in structure, activity, etc., i.e., the humanization of the mouse antibody of the present application is completed.

[0237] The following is described by way of example with the specific sequence of mab 14, using a hlgG4 heavy chain and a kappa type light chain (sequences as follows).

[0238] Human antibody light chain constant region kappa chain:

[0239] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27)

[0240] Human IgG4 heavy chain constant region:

[0241] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 28)

[0242] The preferred sequence of the humanized light chain variable region of the present application is as follows:

[0243] >mab 14-hL1

[0244] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 29)

[0245] > mab14-hL2

[0246] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTVKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 30)

[0247] > mab14-hL3

[0248] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 31)

[0249] > mab14-hL4

[0250] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGAPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 32)

[0251] > mab14-hL5

[0252] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 33)

[0253] > mab14-hL6

[0254] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKTPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 34)

[0255] The present humanized heavy chain variable region preferred sequences:

[0256] >mab14-hH1

[0257] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWMGNIYPSSISTNYNEKFKTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 35)

[0258] >mab14-hH2

[0259] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVKQAPGQGLEWIGNIYPSSISTNYNEKFKTKATLTVDKSTSTVYMEFSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 36)

[0260] >mab14-hH3

[0261] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWMGNIYPSSISTNYNEKFKTRATLTVDTSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 37)

[0262] >mab14-hH4

[0263] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDTSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 38)

[0264] >mab14-hH5

[0265] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 39)

[0266] >mab14-hH6

[0267] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVKQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDTSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 40)

[0268] >mab14-hH7

[0269] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVKQAPGQGLEWIGNIYPSSISTNYNEKFKTKATLTVDTSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSS (SEQ ID NO: 41)

[0270] The humanized sequences of the murine antibody light chains of the present application contain different back mutations, and the number of back mutation sites can be 10 or more, preferably 0-10, such as the sequences listed above. These arbitrary sequences are combined with the constant region sequences of the human antibody light chain constant region kappa chain or lambda chain to obtain the light chain sequences of the antibodies of the present application, such as the kappa type light chain constant region of the light chain of the present application, such as the sequences listed above. Similarly, the heavy chain variable regions used for humanization also have different numbers of back mutations, and the number of back mutation sites can be 10 or more, preferably 0-10, such as the heavy chain variable region sequences listed above. These heavy chain variable region sequences containing different numbers of back mutations are recombined with the constant region sequences of the human IgG1, human IgG2, human IgG3, human IgG4 chain constant region to obtain the heavy chain sequences of the present application, such as the hIgG4 constant region sequence used as an example to illustrate the heavy chain of the present application.

[0271] The humanized antibody sequences of the present application are partially optimized, and the expression amount and activity evaluation results of the expressed antibodies (ELISA detection method of Example 3 of the present application) are as follows.

[0272] Table 13 Humanized antibody sequences of the present application (human kappa light chain, hlgG4 heavy chain constant region as an example)

[0273]

[0274] The above results show that the binding activity of the human-mouse chimeric antibody mab 14c of the present application to hSirpα-vl is almost the same as that of the control molecule Ref 1 (0.093 vs 0.072). The above humanized antibody molecules obtained by combining the light and heavy chain sequences of the human-mouse chimeric antibody sequence mab 14c of the present application with different degrees of humanization in three different combinations all retain the binding activity almost the same as that of the chimeric antibody.

[0275] More preferably, the expression levels of the different combination antibodies between the sequences with different degrees of humanization are significantly different, and the expression levels of the antibodies mab 14-hl, mab 14-hll and mab 14-hl6 are as high as 200 mg / L, among which the expression level of mab 14-hl6 is the highest, reaching 275 mg / L, which is 4.5 times higher than the expression level of the chimeric antibody mab 14c (275 mg / L vs 49 mg / L).

[0276] More particularly, the effect of the humanized antibodies of the present application on blocking the binding of hSirpα (dimer) to hCD47 and the binding of hSirpα to hCD47 (dimer) was detected using the experimental method described in Example 4, and the results are shown in Table 14. The humanized antibodies of the present application retain the property of the chimeric antibody mab 14c that can well block the binding of hSirpα to hCD47. This result shows that the chimeric antibodies and humanized antibodies of the present application not only bind to hSirpα protein, but also effectively block the binding of hSirpα to hCD47.

[0277] Table 14 Activity of humanized antibodies of the present application (human kappa light chain, hlgG4 heavy chain constant region as an example)

[0278]

[0279] The amino acid sequences of the light and heavy chains (including the constant region) of the humanized antibodies (partially representative molecules) are shown below.

[0280] Amino acid sequence of humanized mab 14-h5 antibody:

[0281] Light chain:

[0282] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTVKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42)

[0283] Heavy chain:

[0284] QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 43)

[0285] Humanized mab 14-h12 antibody amino acid sequence:

[0286] Light chain:

[0287] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGAPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 45)

[0288] Humanized mab14-h13 antibody amino acid sequence:

[0289] Heavy chain:

[0290] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGAPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 45)

[0291] Humanized antibody mab14-h13 heavy chain sequence is the same as SEQ ID NO: 43.

[0292] Humanized mab14-h14 antibody amino acid sequence:

[0293] Heavy chain:

[0294] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGAPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 45)

[0295] The humanized antibody mab14-h14 heavy chain sequence is identical to SEQ ID NO: 43.

[0296] The humanized mab14-h15 antibody amino acid sequence is:

[0297] Light chain:

[0298] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKTPKLLIYFTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 47)

[0299] The humanized antibody mab14-h15 heavy chain sequence is identical to SEQ ID NO: 43.

[0300] The humanized mab14-h16 antibody amino acid sequence is:

[0301] Light chain:

[0302] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 48)

[0303] The humanized antibody mab14-h16 heavy chain sequence is identical to SEQ ID NO: 43.

[0304] Example 9 Comprehensive evaluation of the binding activity of the humanized anti-human Sirpa antibodies of the present application, preferably antibodies

[0305] To further evaluate the binding activity of the humanized antibodies of the present application, the humanized preferred antibody mab 14-h16 of the present application was taken as an example, and it and the control antibody Ref were evaluated in parallel / repeated binding activity (ELISA) evaluation. The binding activity of the antibodies with hSirpα-Vl, hSirpα-V2, hSirpβ, hSirpγ and different polymorphism cyno Sirpα was detected, and the experimental method was the same as that of Example 3 above, and the results are shown in Table 15 below and Figure 2 and Figure 3 .

[0306] Table 15 Binding activity (EC 50 , nM) of the antibodies of the present application with Sirpα

[0307]

[0308] ND, no binding signal detected, i.e. no binding; weak binding, EC 50 higher than 10 nM; weak binding, EC 50 between 2-10 nM.

[0309] The above results show that the mouse anti-human Sirpα antibody and the humanized anti-human Sirpα antibody unexpectedly discovered by the present application are a very specific and new antibody. The antibody, unlike Ref 1, can simultaneously bind human Sirpα-Vl, V2 and various cyno Sirpα (polymorphism), and does not bind Sirpβ.

[0310] Furthermore, the new antibody of the present application is also significantly different from Ref 2. Ref 2 can also simultaneously bind Sirpα-Vl and V2, but the binding activity is weaker than that of the antibody of the present application. The binding activity of the antibody mab 14-h16 of the present application with Vl is 1.4 times stronger than that of Ref 2 (0.065 nM vs 0.159 nM in the above table), and the binding with V2 is 2.9 times stronger than that of Ref 2 (0.094 nM vs 0.37 nM). More significantly different is that Ref 2 has relatively strong binding with Sirpβ and Sirpγ, with EC 50 values of 0.167 nM and 1.46 nM, respectively. The antibody of the present application only has weak or no binding with Sirpβ and Sirpγ. This unique selectivity (specific binding with Sirpα but not with Sirpβ and Sirpγ) of the present application makes it have outstanding advantages in the development process as a drug in clinic, i.e. it can avoid the safety problems caused by non-targeting.

[0311] In addition, Ref 2 can bind cyno Sirpα polymorphisms 5 and 6. This is also different from the antibody of the present application.

[0312] These features indicate that the antibody of the present application and Ref1, Ref2 have different epitopes.

[0313] To further evaluate the cell binding activity of the antibodies of the present application, blocking the activity of Sirpα and the corresponding ligand CD47, the present application takes the humanized antibody mab14-h16 as an example, and uses a human Sirpα+high expression cell line to detect its binding activity with a human Sirpα high expression cell line by plate ELISA, and the detection method is as shown in Example 2. The ability of the humanized antibody of the present application to block the binding activity of human Sirpα and CD47 is detected by the method in Example 4, and the binding of the humanized antibody to human T cells is detected by the method in Example 5. The results are shown in Table 16a.

[0314] In addition, the blocking of the binding of human Sirpα-V2 and human CD47 by the antibodies of the present application is further detected. Specifically, the human Sirpα-V2-his recombinant protein is diluted to a concentration of 2 μg / ml with PBS buffer at pH 7.4, and added to a 96-well enzyme-labeled plate (Corning, CLS3590-100EA) at a volume of 50 μl / well, and placed in a 37°C incubator for 2 hours. After discarding the liquid, 230 μl / well of 5% skimmed milk (Guangming skimmed milk powder) blocking solution diluted with PBS is added, and incubated in a 37°C incubator for 3 hours or placed at 4°C overnight (16-18 hours) for blocking. After discarding the blocking solution, the plate is washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), 25 μl / well of the antibody to be tested is added, starting at 100 μg / ml, and diluted by 3 times in gradient, and 25 μl / well of biotin labeled CD47-hFc or CD47-his at a concentration of 4 μg / ml is added, mixed well, and incubated in a 37°C incubator for 1 hour. After washing the plate 5 times, 50 μl / well of streptavidin-HRP secondary antibody (genscript, M00091) diluted 1:1000 is added, and incubated at 37°C for 1 hour. After washing 5 times with PBST, 50 μl of TMB (Surmodic Cat#TTMB-1000-01) is added to each well for color development, and 50 μl / well of 1M H2SO4 is added to stop the reaction. The reading is performed by an enzyme-labeled instrument (MultiskanGO Thermo model 51119200), and the data analysis is performed by Graphpad prism 5. The results are shown in Table 16b.

[0315] Table 16a Evaluation of cell binding activity and blocking activity of antibodies of the present application

[0316] Antibody sample mab14-h16 Ref1 Ref2 T cell binding activity (percent binding %) 1.95 1.79 47.6 hSirpα-v1+ cell line binding activity (EC50, nM) 0.124 0.084 0.106 Block hSirpα (dimer) binding to CD47 (IC50, nM) 4.81 3.74 4.79 Block hSirpα binding to CD47 (dimer), IC50 (nM) 5.28 4.16 5.44

[0317] Table 16b Evaluation of the activity of the antibodies of the present application in blocking the binding of human Sirpα-V2 to human CD47

[0318] Antibody sample mab14-h16 Ref1 Ref2 Block hSirpα-V2 binding to hCD47 (IC50, nM) 2.39 ND 1.29 Block hSirpα-V2 binding to human CD47 (dimer), IC50 (nM) 6.29 ND 3.09

[0319] ND: no detectable blocking signal.

[0320] The results of the above table show that the humanized antibodies of the present application and the preferred antibody mab14-h16 do not bind to human T cells (the negative control bound 1.67% in this assay and mab14-h16 bound 1.95% which is close to the background level). The binding activity to hSirpα-v1+ cell line, the blocking activity of hSirpα-v1 and human CD47 binding activity are close to the controls Ref1, Ref2, and can effectively block the binding of hSirpα-V2 and human CD47.

[0321] The data of the above examples show that the mouse anti-human Sirpα antibodies, humanized anti-human Sirpα antibodies of the present application unexpectedly found not only can bind to human Sirpα-V1, human Sirpα-V2 at the same time, and the binding activity to human Sirpα-V1, human Sirpα-V2 is stronger, can bind to multiple cyno Sirpα (polymorphism), and do not bind to human Sirpβ and Sirpγ.

[0322] In addition, the antibodies of the present application have very good blocking activity of human Sirpα (including Sirpα-V1 and Sirpα-V2) and human CD47 binding, so that the antibodies can be developed as new drugs, targeting Sirpα and CD47 binding to achieve the purpose of treating tumors.

[0323] These outstanding features make the antibodies of the present application show unique advantages in clinical practice, which can be reflected in the clinical practice that more patient groups (people expressing V1 and people expressing V2) can be targeted. Do not bind to Sirpβ and Sirpγ, and T cells, have very good specificity, avoid the side effects caused by non-targeting in clinical practice. At the same time, the antibodies of the present application can strongly bind to multiple cyno Sirpα, and can choose cyno primate in preclinical safety evaluation studies, which provides convenience for preclinical studies.

[0324] Example 10 Humanized anti-human Sirpα antibody PTM of the present application

[0325] By means of MOE (molecular operating environment), https: / / www.chemcomp.com / Products.htm; Schrodinger, https: / / www.schrodinger.com / ; or DS (Discovery Studio) and other computer analysis software, the antibody of the present application is subjected to post-translational modification (PTM) site analysis. The results show that only M4 among M4, C23, W35, C88, W96 of the light chain and C22, W33, W36, W47, M81, C96, W112 of the H chain of the preferred antibody of the present application is a low-risk oxidation site, and the others are non-oxidation hotspots. Except for the slight deamidation risk of N92 of the light chain, there is no other deamidation site or hotspot. There is no N-glycosylation hotspot, no aspisomerization site or hotspot in the whole sequence. Therefore, the humanized sequence of the present application is a preferred sequence for PTM analysis.

[0326] Example 11 Design of bispecific antibodies against Sirpα target

[0327] Based on the anti-Sirpα antibodies discovered above, the present application designs a plurality of bispecific antibodies. The general formula of the designed bispecific antibodies is as follows.

[0328] Table 17 Design of bispecific antibodies based on anti-Sirpα antibodies of the present application (general formula 1)

[0329] Protocol Sequence containing light chain Sequence containing heavy chain 1 T2(scFv) n1 T1VL-LC-T2(scFv) n2 ]]> T2(scFv) n3 T2(scFv) n4 ]]> 2 [T1(scFv) n1 -T2VL-LC - T1(scFv) n2 ]]> [T1(scFv) n3 -T2VH-HC-T1(scFv) n4 ]]> 3 [T2(scFv) n1 -T1VL-LC - T1(scFv) n2 ]]> [T2(scFv) n3 -T1VH-HC-T1(scFv) n4 ]]> 4 [T1(scFv) n1 -T2VL-LC - T2(scFv) n2 ]]> [T1(scFv) n3 -T2VH-HC-T2(scFv) n4 ]]>

[0330] In Table 17, the sequence containing the light chain refers to a sequence that includes the light chain sequence and can also include an scFv connected to the light chain sequence; the sequence containing the heavy chain refers to a sequence that includes the heavy chain sequence and can also include an scFv connected to the heavy chain sequence. Among them, T1 represents the first protein functional region against target 1 (such as Sirpα), and T2 represents the second protein functional region against target 2 (non-Sirpα). T1 (scFv) represents the scFv sequence of the antibody against target 1; T2 (scFv) represents the scFv sequence against target 2.

[0331] (scFv) n1 , (scFv) n2 , (scFv) n3 , (scFv) n4n1, n2, n3, n4 are natural numbers, which can be 0, 1, 2, 3, etc. In specific embodiments of the present application, at least one of n1, n2, n3, n4 is 1, and the rest are 0. VL represents the antibody light chain variable region sequence against target 1 or 2; VH represents the antibody heavy chain variable region sequence against target 1 or 2. LC represents the constant region sequence of light chain (κ or λ), preferably the human light chain constant region sequence; HC represents the heavy chain, including the constant region sequence of IgG1, IgG2, IgG3, IgG4 (abbreviated as HC-IgG1, HC-IgG2, HC-IgG3, HC-IgG4), preferably the human heavy chain constant region sequence (HC-hIgG). When the C-terminal of the heavy chain constant region is connected with the scFv or other protein sequence, the C-terminal last amino acid K can be mutated, preferably mutated to A. Thus, in Scheme 1, T1 is an immunoglobulin, and T2 is an scFv; in Scheme 2, T2 is an immunoglobulin, and T1 is an scFv; the scFv targets the same target; in Schemes 3 and 4, the scFv at both ends targets two different targets.

[0332] In Table 17, the scFv is a light chain variable region-linker-heavy chain variable region, and the N-terminal of the light chain variable region or the C-terminal of the heavy chain variable region is connected to the C-terminal or N-terminal of the light chain and / or heavy chain of the immunoglobulin via a linker, respectively; or the scFv is a heavy chain variable region-linker-light chain variable region, and the N-terminal of the heavy chain variable region or the C-terminal of the light chain variable region is connected to the C-terminal or N-terminal of the light chain and / or heavy chain of the immunoglobulin via a linker, respectively.

[0333] It should be noted that when the scFv is a light chain variable region-linker-heavy chain variable region, the C-terminal of the light chain variable region is connected to the linker, and the linker is connected to the N-terminal of the heavy chain variable region, thereby exposing the N-terminal of the light chain variable region and the C-terminal of the heavy chain variable region of the scFv, so that they can be connected to the light chain and / or heavy chain of the immunoglobulin via a linker. In the present application, when it is connected to the light chain of the immunoglobulin, the C-terminal of the heavy chain variable region of the scFv is preferably connected to the N-terminal of the heavy chain of the immunoglobulin via a linker in some specific embodiments; when it is connected to the heavy chain of the immunoglobulin, the N-terminal of the light chain variable region of the scFv is preferably connected to the C-terminal of the heavy chain of the immunoglobulin.

[0334] When the scFv is a heavy chain variable region-linker-light chain variable region, it is connected in a way that the N-terminus of the light chain variable region is connected to the linker, which is then connected to the C-terminus of the heavy chain variable region, thus exposing the C-terminus of the scFv light chain variable region and the N-terminus of the heavy chain variable region, so that they can be connected to the light chain and or heavy chain of the immunoglobulin through the linker. In this case, when it is connected to the light chain of the immunoglobulin, in some specific embodiments, it is preferred to use the C-terminus of the light chain variable region of the scFv to connect to the N-terminus of the heavy chain of the immunoglobulin; when it is connected to the heavy chain of the immunoglobulin, in some specific embodiments, it is preferred to use the N-terminus of the heavy chain variable region of the scFv to connect to the C-terminus of the heavy chain of the immunoglobulin.

[0335] The linker is preferably (G4S) m , and the m is preferably an integer between 0 and 10. Further preferably, the linker is (Gly-Gly-Gly-Gly-Ser)3, and / or the number of scFv is one pair, symmetrically connected to the C-terminus and or N-terminus of the light chain and / or heavy chain of the immunoglobulin.

[0336] The sequences of the antibodies for each of the target points involved in the above-mentioned bispecific design, except for the anti-Sirpα antibody sequences described in the present application, are from already published antibody sequences. Including anti-PD-1 antibodies Nivolumab / Opidivo (referred to as Nivo) and Pembrolizumab / Keytruda (referred to as Pem). The sequences of Nivolumab and Pembrolizumab, etc. can be found in public resources such as www.drugbank.ca.

[0337] Example 12 Antigen-antibody binding (ELISA) experiment

[0338] The self-expressed human PD-1, Sirpa, and other antigens of the application are diluted to a concentration of 2 pg / ml with PBS buffer at pH 7.4 according to different assays, and added to a 96-well enzyme-labeled plate (Corning, CLS3590-100EA) at a volume of 50 mI / well, and placed in a 37°C incubator for 2 hours. After discarding the liquid, 200 mI / well of 5% skimmed milk (Shanghai Biotechnology Engineering Co., Ltd., A600669-0250) blocking solution diluted with PBS is added, and incubated in a 37°C incubator for 3 hours or placed at 4°C overnight (16-18 hours) for blocking. After discarding the blocking solution, the plate is washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), 50 mI / well of 1% BSA 5-fold serially diluted test antibody is added, and incubated at 37°C for 1 hour, the plate is washed 5 times with PBST, 50 mI / well of 1:2500 diluted HRP-labeled secondary antibody (Jackson Immuno Research, 115-035-003) is added, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 mI / well of TMB color developing substrate (KPL, 52-00-03) is added, and incubated at room temperature for 5-10 min, 50 mI / well of 1 M H2SO4 is added to terminate the reaction, and the absorbance value is read at 450 nm with a MULTISKAN Go enzyme-labeled instrument (ThermoFisher, 51119200), and the EC 50 .

[0339] Example 13 Antibody Blocking Antigen Antibody Binding (Blocking) Experiment

[0340] The antigen PD-1, Sirpα expressed according to the method in Example 1 was diluted to a concentration of 2 μg / ml with PBS buffer at pH 7.4, and added to a 96-well enzyme plate (Corning, CLS3590-100EA) at a volume of 50 μl / well, and incubated at 37°C for 2 hours. After discarding the liquid, 200 μl / well of blocking solution of 5% skimmed milk (Shanghai Genechem Biotech Co., Ltd., A600669-0250) diluted with PBS was added, and incubated at 37°C for 3 hours for blocking. After discarding the blocking solution, the plate was washed 5 times with PBST (pH 7.4 PBS containing 0.05% tweeen-20), and 25 μl of the sample to be tested diluted 3 times in succession with 1% BSA and 25 μl of biotin-labeled ligand (CD47-his / CD47-hFc, PD-L1, etc., expressed and purified according to the application) at a final concentration of 1 μg / ml or 12 ug / ml were added to each well, and incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, and 50 μl / well of HRP-labeled secondary antibody (Genscript Biotech Co., Ltd., M00091) diluted 1:1000 was added, and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, 50 μl / well of TMB color developing substrate (KPL, 52-00-03) was added, and incubated at room temperature for 5-10 min, and the reaction was stopped by adding 50 μl / well of 1M H2SO4, and the absorbance value was read at 450 nm with a MULTISKAN Go enzyme reader (ThermoFisher, 51119200), and the IC 50 .

[0341] The biotin labeling kit was Biotin Labeling Kit-NH2, purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., product number LK03. The operation method was performed according to the instructions, and the labeled antibody was detected for concentration with a Multiskan GO (ThermoFisher) enzyme reader before use.

[0342] Example 14 Design and activity evaluation of bispecific antibodies against Sirpα and PD-1 double targets

[0343] The application designed bispecific antibodies of different sequence structures against the two targets of Sirpα and PD-1, as shown in the following table.

[0344] Table 18 Bispecific antibodies designed against Sirpα and PD-1 double targets

[0345]

[0346] *: The κ chain indicates that the light chain is a κ type light chain constant region of human IgG. #: When the C-terminal of IgG4 is connected to the linker, the last amino acid K is mutated to A. The following heavy chain C-terminal introduces the design of scFv, and the last K is mutated to A.

[0347] According to the cloning, expression, and purification method described in Example 1 of this invention, the above-mentioned bispecific antibodies were cloned, expressed, and purified respectively. The binding activity of these designed bispecific molecules to human Sirpα and PD-1 was detected by the methods in Examples 12 and 13 above. It was found that LB501, LB502, LB503, LB504, and LB506 could retain the binding activity to the two target antigens. The results are shown in the table below.

[0348] Table 19 Evaluation of the binding activity of bispecific antibodies designed targeting both Sirpα and PD-1.

[0349]

[0350] #: The value in parentheses represents the binding activity (EC) of the monoclonal antibody targeting the same target under the same experimental conditions. 50 *: Under the same experimental conditions, the binding activity (ECg) of bispecific antibodies and their corresponding monoclonal antibodies is... 50 The ratio is the ratio of the bispecific antibody to the target. A higher ratio indicates a greater reduction in the binding affinity of the designed bispecific antibody to the single target. For example, a ratio of 2 indicates that the binding activity of the designed bispecific antibody to the target is reduced by half compared to the corresponding monoclonal antibody. A ratio within 2 (within the experimental error range) indicates that the binding activity is not affected.

[0351] The table above shows the bispecific molecules designed by the scFv of the anti-Sirpα antibody mab14-h16 of the present invention at the N-terminus and C-terminus of the heavy chain of the PD-1 antibody Pem, or at the N-terminus and C-terminus of the heavy chain of the PD-1 antibody Nivo, or the bispecific molecules designed by the scFv of the PD-1 antibody Pem and the anti-Sirpα antibody mab14-h16 of the present invention.

[0352] The results show that the same Sirpα antibody and scFv, different positions designed into bispecific, the effect on Pem activity is much smaller than Nivo, such as LB501, LB502, LB503 and LB504 (scFv connected on Pem), the binding activity of two target points is close to the corresponding single antibody, and compared with scFv connected on Nivo, only LB506 is close to the binding activity of two target points and the corresponding single antibody molecule. It is indicated that different positions of Sirpα antibody scFv have little effect on the activity of Pem, and the effect on the activity of Nivo is different. Similarly, the scFv of Pem connected to the N-terminal of the heavy chain of the anti-Sirpα antibody mab14-h16 of the application (LB507) and the C-terminal of the heavy chain (LB508) to form a bispecific antibody has different binding activities of the antibody, the binding activity of LB507 to Sirpα is weakened by 1 times compared with the single antibody, and the binding activity to PD-1 is close to Pem, but the binding activity of LB508 to two target points is significantly decreased.

[0353] The above data of the designed bispecific antibodies of the application show that the same Sirpα antibody (of the application) and scFv, the same design method, but the sequence of PD-1 antibody is different, and the activity of the designed bispecific antibody molecules is greatly different. The same Sirpα antibody (of the application) scFv and PD-1 antibody, but the position of Sirpα antibody (of the application) scFv is different, and the activity of the designed bispecific antibody molecules is greatly different. The same PD-1 antibody scFv and Sirpα antibody (of the application), but the position of PD-1 antibody scFv is different, and the activity is also greatly different.

[0354] The data show that the designed bispecific antibodies based on the Sirpα antibody sequence of the application have different sequences, different positions of scFv and antibodies, and different activities. The appropriate position and appropriate sequence design can obtain bispecific antibodies with good activity to double target points. The structure of these bispecific antibodies is similar to the conventional IgG, and the Fc is complete, which is referred to as Sequence-based IgG like bispecific antibody format in the application, i.e., sequence-specific IgG-like bispecific antibody (SBody). These bispecific antibody molecules have a complete Fc as normal antibodies, so that the purification process can be carried out according to the normal antibodies, and thus the process is simple, and has the advantage of low production cost.

[0355] The SBody with the above-mentioned double target point activity was respectively evaluated for the function (blocking antigen and corresponding ligand binding experiment) of two target points, and the results are shown in the following table.

[0356] Table 20 Evaluation of the functional activity of the bispecific antibody designed for Sirpα and PD-1 double target points

[0357]

[0358] #: The value in parentheses represents the IC50 value for the monoclonal antibody blocking antigen-ligand binding activity at the same target under the same experimental conditions. 50 *: IC 50 Changing the fold, i.e., the IC50 ratio of the bispecific antibody and the corresponding monoclonal antibody (control antibody). 50 The ratio. The larger the ratio, the more the designed bispecific antibody's functional activity against the single target is weakened. For example, a ratio of 2 indicates that the designed bispecific antibody's functional activity against the target is weakened by half compared to the corresponding monoclonal antibody. A ratio within 2 is within the experimental error range, meaning the activity is not affected. ND: No inhibition of Sirpα and Daudi cell binding activity was detected in this molecule.

[0359] The above functional activity results indicate that the bispecific antibodies (SBody) designed in this invention exhibit changes in activity that block the binding of antigens to their corresponding ligands consistent with changes in binding activity. For example, LB507 shows a slight decrease in activity binding to human Sirpα and human PD-1, as well as a slight decrease in activity blocking the binding of human Sirpα and human CD47 and the binding of human PD-1 to human PD-L1 (compared to the corresponding monoclonal antibodies, the fold changes are 2.46, 2.59, and 2.20, respectively). Other designs, LB501, LB502, LB503, LB504, and LB506, all retain functional activity against both targets.

[0360] To evaluate the expression level of the bispecific antibody SBody of the present invention, the SBody was transiently transfected in the same expression system (293F cells) using the same method, purified with conventional Protein A, and the expression levels were obtained. The results are shown in the table below.

[0361] Table 21 Evaluation of expression levels of bispecific antibodies designed targeting Sirpα and PD-1.

[0362] Antibody number Expression amount (mg / L) Antibody number Expression amount (mg / L) LB501 2.24 LB504 7.35 LB502 2.43 LB506 44.29 LB503 3.83

[0363] The above results show that the expression yield of the SBody designed against Sirpα and PD1 is quite different. Based on the above data analysis, the expression yield of the SBody with the same design mode, the same Sirpα antibody scFv and different PD-1 antibody sequences is different. The expression yield of the SBody corresponding to Nivo is 5 times higher than that of Pem, and even 17 times. For example, the yield of LB506 is 17 times higher than that of LB502 (44.29 / 2.43); the yield of LB506 is 5 times higher than that of LB504 (44.29 / 7.35). The expression yield of the SBody with the same design mode, the same PD-1 sequence and the same Sirpα antibody scFv sequence but different scFv positions is also different by more than 2 times. For example, LB504 (7.35 mg / L) vs LB501 (2.24 mg / L) vs LB502 (2.43 mg / L).

[0364] These data show that the SBody designed by the anti-Sirpα antibody mab14-h16 and the PD1 antibody of the application is not only sequence-specific in activity and function, but also in expression yield.

[0365] The partial sequences of the SBody designed by the anti-Sirpα antibody mab14-h16 and the PD1 antibody of the application are as follows:

[0366] The sequence of the light chain of LB501 is as follows:

[0367] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50)

[0368] LB501 heavy chain sequence (Pem heavy chain):

[0369] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 51)

[0370] LB502 light chain sequence (Pem light chain):

[0371] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 52)

[0372] LB502 heavy chain sequence:

[0373] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 53)

[0374] LB503 light chain sequence:

[0375] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 54)

[0376] LB503 heavy chain sequence (Pem heavy chain): identical to SEQ ID NO: 51.

[0377] LB504 light chain sequence (Pem light chain): identical to SEQ ID NO: 52.

[0378] LB504 heavy chain sequence:

[0379] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGAGGGGSGGGGSGGGGSGGGGQVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 55)

[0380] LB506 heavy chain sequence (Nivo heavy chain):

[0381] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56)

[0382] LB506 heavy chain sequence:

[0383] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGAGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 57)

[0384] In summary, the present application data show that the inventors unexpectedly discovered an anti-human Sirpα antibody through innovative screening, which has good binding activity with Sirpα, can simultaneously bind human Sirpα-V1 and human Sirpα-V2, and has good binding activity with various polymorphic proteins of non-human primate cynomolgus Sirpα. It can effectively block the binding of human Sirpα and human CD47. It has better activity than the current clinical antibodies (control antibodies Ref1 and Ref2); and does not bind human Sirpβ and human Sirpγ, nor human T cells, has good selectivity, can avoid off-target effects caused by clinical non-targeting, and can more effectively avoid side effects. In addition, the sequence of the molecule itself has low PTM risk. After humanization, the antibody has high expression, which provides convenience for downstream production and process, and saves cost. In addition, the bispecific antibody designed based on the Sirpα antibody sequence of the present application can retain the functional activity of the bispecific antibody, and the binding activity to the two target points is close to that of the corresponding monoclonal antibody. The activity of blocking the binding of the antigen to the corresponding ligand is also consistent with the binding activity of the corresponding monoclonal antibody. These bispecific antibodies (referred to as SBody in the present application) have a structure similar to conventional IgG, have a complete Fc as normal antibodies, so that the purification process can be carried out according to normal antibodies, and thus the process is simple, and has the advantage of low production cost. The unique characteristics of the antibody of the present application make it more suitable for the development of antibody drugs targeting human Sirpα target points, and can be administered alone or in combination as a candidate drug. In particular, it provides a new and even better choice for the combination of PD-1 antibody for tumor treatment, and the preferred bispecific antibody of the present application provides another choice for multi-target point treatment of tumors. SEQUENCE LISTING <110> Shanghai Jianshen Biomedical Technology Co., Ltd. <120> Antibodies or antigen-binding fragments thereof targeting Sirpα and preparation and application thereof <130> P19014766C <160> 57 <170> PatentIn version 3.5 <210> 1 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> PCR primer SI-2F <400> 1 taaacggatc tctagcgaat tcatggagcc cgccggcccg gcccccg 47 <210> 2 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> PCR primer SI-2R <400> 2 cggccttgcc ggcctcgagc ggccgctgtc tgattcggac gaggtagag 49 <210> 3 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> RB3-3 sequence <400> 3 Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Asp Ser Ala Thr Leu Asn Cys Thr Val Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr His Gln Lys Glu Gly His Phe Pro Arg Val Thr Ser Val Ser 50 55 60 Glu Ser Thr Lys Arg Asn Asn Met Asp Phe Ser Ile His Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Val Glu Val Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg 115 120 125 Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe 130 135 140 Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu 145 150 155 160 Ser Asp Phe Gln Thr Asn Val Asp Pro Ala Gly Lys Ser Val Ser Tyr 165 170 175 Ser Ile Arg Ser Thr Ala Arg Val Val Leu Thr Arg Arg Asp Val His 180 185 190 Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro 195 200 205 Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Phe 210 215 220 Leu Glu Val Thr Gln Gln Ser Met Arg Ala Asp Asn Gln Val Asn Val 225 230 235 240 Thr Cys Gln Val Thr Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp 245 250 255 Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser Ala Leu Pro 260 265 270 Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu Leu Val Asn 275 280 285 Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His 290 295 300 Asp Gly Gln Pro Ala Val Asn Lys Ser Phe Ser Val Lys Val Ser Ala 305 310 315 320 His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Thr 325 330 335 Asn Glu Arg Asn 340 <210> 4 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> RB3-5 sequence <400> 4 Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Asp Ser Ala Thr Leu Asn Cys Thr Val Ser Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr Asn Leu Lys Glu Gly His Phe Pro Arg Val Thr Pro Val Ser 50 55 60 Asp Pro Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Val Glu Leu Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg 115 120 125 Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe 130 135 140 Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu 145 150 155 160 Ser Asp Val Gln Thr Asn Val Asp Pro Ala Gly Lys Ser Val Ser Tyr 165 170 175 Ser Ile Arg Ser Thr Ala Arg Val Leu Leu Thr Arg Arg Asp Val His 180 185 190 Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro 195 200 205 Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Phe 210 215 220 Leu Glu Val Thr Gin Gin Ser Met Arg Ala Asp Asn Gin Val Asn Val 225 230 235 240 Thr Cys Gin Val Thr Lys Phe Tyr Pro Gin Arg Leu Gin Leu Thr Trp 245 250 255 Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser Ala Leu Pro 260 265 270 Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu Leu Val Asn 275 280 285 Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gin Val Glu His 290 295 300 Asp Gly Gin Pro Ala Val Asn Lys Ser Phe Ser Val Lys Val Ser Ala 305 310 315 320 His Pro Lys Glu Gin Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Thr 325 330 335 Asn Glu Arg Asn 340 <210> 5 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> RB6-1 sequence <400> 5 Glu Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Thr Leu Asn Cys Thr Ala Thr Ser Leu Ile Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu 35 40 45 Ile Tyr His Gln Lys Glu Gly His Phe Pro Arg Val Thr Pro Val Ser 50 55 60 Asp Pro Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Val Glu Leu Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg 115 120 125 Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe 130 135 140 Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu 145 150 155 160 Ser Asp Phe Gin Thr Asn Val Asp Pro Ala Gly Lys Ser Val Ser Tyr 165 170 175 Ser lie Arg Ser Thr Ala Arg Val Val Leu Thr Arg Arg Asp Val His 180 185 190 Ser Gin Val lie Cys Glu Val Ala His Val Thr Leu Gin Gly Asp Pro 195 200 205 Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala lie Arg Val Pro Pro Phe 210 215 220 Leu Glu Val Thr Gin Gin Ser Met Arg Ala Asp Asn Gin Val Asn Val 225 230 235 240 Thr Cys Gin Val Met Lys Phe Tyr Pro Gin Arg Leu Gin Leu Thr Trp 245 250 255 Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser Ala Leu Pro 260 265 270 Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu Leu Val Asn 275 280 285 Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gin Val Glu His 290 295 300 Asp Gly Gin Pro Ala Val Asn Lys Ser Phe Ser Val Lys Val Ser Ala 305 310 315 320 His Pro Lys Glu Gin Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Thr 325 330 335 Asn Glu Arg Asn 340 <210> 6 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> RB6-2 sequence <400> 6 Glu Glu Glu Leu Gin Val lie Gin Pro Glu Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Thr Leu Asn Cys Thr Ala Thr Ser Leu lie Pro 20 25 30 Val Gly Pro lie Gin Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu 35 40 45 lie Tyr Ser Gin Lys Glu Gly His Phe Pro Arg Val Thr Pro Val Ser 50 55 60 Asp Pro Thr Lys Arg Asn Asn Met Asp Phe Ser lie Arg lie Ser Asn 65 70 75 80 lie Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Val Glu Leu Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg 115 120 125 Ala Thr Ala Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe 130 135 140 Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu 145 150 155 160 Ser Asp Phe Gln Thr Asn Val Asp Pro Ala Gly Lys Ser Val Ser Tyr 165 170 175 Ser Ile Arg Ser Thr Ala Arg Val Val Leu Thr Arg Arg Asp Val His 180 185 190 Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro 195 200 205 Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Phe 210 215 220 Leu Glu Val Thr Gln Gln Ser Met Arg Ala Asp Asn Gln Val Asn Val 225 230 235 240 Thr Cys Gln Val Thr Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp 245 250 255 Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Met Ala Ser Ala Leu Pro 260 265 270 Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Leu Leu Val Asn 275 280 285 Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His 290 295 300 Asp Gly Gln Pro Ala Val Asn Lys Ser Phe Ser Val Lys Val Ser Ala 305 310 315 320 His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Thr 325 330 335 Asn Glu Arg Asn 340 <210> 7 <211> 501 <212> DNA <213> Artificial Sequence <220> <223> Mouse monoclonal antibody mab14 light chain variable region base sequence <400> 7 taatggtgtc cctcagctca gttccttggt ctcctgttgc tctgttttca aggtaccaga 60 tgtgatatcc agatgacaca gactacatcc tccctgtctg cctctctctg ggagacagag tc 120 accatcagtt gcagggcaag tcaggacatt aggaattatt taaactggta tcagcagaag 180 ccagatggaa ctgttaaact cctgatctac ttcacatcaa cattacactc aggagtccca 240 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggaa 300 caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgtg gacgttcggt 360 ggaggcacca agctggaaat caaacgggct gatgctgcac caactgtatc catcttccca 420 ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctg aacaactcta 480 ccccaaagac atcaaggacc t 501 <210> 8 <211> 550 <212> DNA <213> Artificial Sequence <220> <223> Mouse monoclonal antibody mab14 heavy chain variable region base sequence <400> 8 tcatgggatg gagctgtatc atgttctttt tggtagccgc agctacaggt gtccactccc 60 aggtccatct gcagcagcct ggggctgagc ttgtgaagcc tggggcttca gtgaagttgt 120 cctgcaaggc ttctggctac aatttcaaca tctactggat aaattgggtg aagcagaggc 180 ctggacaagg ccttgagtgg attggaaata tttatcctag tagtattagt actaactaca 240 atgagaagtt caagacgaag gccacactga ctgtagacaa atcctccaac acagtctaca 300 tgcagttcag cagcctgaca tctgaggact ctgcggtcta ttattgtgcg cgatcggagg 360 gaacttacta tggtggtcgc tacgaggggg actggtttgg ttactggggc caagggactc 420 tggtcactgt ctctgcagcc aaaacaacac ccccatcagt ctatccactg gcccctgggt 480 gtggagatac aactggttcc tccgtgactc tgggatgcct ggtcaagggc tactgccgag 540 tcgaagttcc 550 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Mouse monoclonal antibody mab14 light chain variable region amino acid sequence <400> 9 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gin 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Murine monoclonal antibody mab 14 heavy chain variable region amino acid sequence <400> 10 Gln Val His Leu Gin Gin Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Val Tyr 65 70 75 80 Met Gin Phe Ser Ser Leu Thr Ser Gin Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gin Asp He Arg Asn Tyr Leu Asn 100 105 110 Phe Gin Ser Trp Gin Gin Gin Thr Leu Val Thr Val Ser Ala 115 120 125 <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CCG-defined light chain CDR1 <400> 11 Arg Ala Ser Gin Asp He Arg Asn Tyr Leu Asn 1 5 10 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CCG-defined light chain CDR2 <400> 12 Phe Thr Ser Thr Leu His Ser 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CCG-defined light chain CDR3 <400> 13 Gln Gin Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CCG-defined heavy chain CDR1 <400> 14 Gly Tyr Asn Phe Asn Ile Tyr Trp Ile Asn 1 5 10 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CCG-defined heavy chain CDR2 <400> 15 Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CCG-defined heavy chain CDR3 <400> 16 Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp Phe Gly 1 5 10 15 Tyr <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Kabat-defined heavy chain CDR1 <400> 17 Ile Tyr Trp Ile Asn 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 as defined by AbM <400> 18 Asn Ile Tyr Pro Ser Ser Ile Ser Thr 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Chothia's definition of heavy chain CDR1 <400> 19 Gly Tyr Asn Phe Asn Ile Tyr 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Chothia's definition of heavy chain CDR2 <400> 20 Tyr Pro Ser Ser Ile 1 5 <210> twenty one <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Contact defines the light chain CDR1 <400> twenty one Arg Asn Tyr Leu Asn Trp Tyr 1 5 <210> twenty two <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Contact-defined light chain CDR2 <400> 22 Lys Leu Leu Ile Tyr Phe Thr Ser Thr Leu His 1 5 10 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Contact-defined light chain CDR3 <400> 23 Gln Gln Gly Asn Thr Leu Pro Trp 1 5 <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Contact-defined heavy chain CDR1 <400> 24 Asn Ile Tyr Trp Ile Asn 1 5 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Contact-defined heavy chain CDR2 <400> 25 Trp Ile Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr 1 5 10 <210> 26 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Contact-defined heavy chain CDR3 <400> 26 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 1 5 10 15 Phe Gly <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human antibody light chain constant region kappa chain <400> 27 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 28 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 heavy chain constant region <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gin Gin Gin Gin Gin Gin 290 295 300 Cys Ser Val Met His Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 305 310 315 320 Leu Ser Leu Ser Leu Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 325 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mab14-hL1 <400> 29 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Gly Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mab14‑hL2 <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mab14-hL3 <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mab14-hL4 <400> 32 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mab14-hL5 <400> 33 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> mab14-hL6 <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 35 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH1 <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH2 <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH3 <400> 37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 38 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH4 <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 39 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH5 <400> 39 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 40 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH6 <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 41 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> mab14-hH7 <400> 41 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab14-h5 antibody amino acid sequence light chain <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 43 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab14-h5 antibody amino acid sequence heavy chain <400> 43 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 130 135 140 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 195 200 205 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 210 215 220 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 325 330 335 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn Gin 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 435 440 445 Leu Ser Leu Gly Lys 450 <210> 44 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab 14-h12 antibody amino acid sequence light chain <400> 44 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Gly Thr Pro Lys Leu Leu lie 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 45 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab14-h13 antibody amino acid sequence light chain <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 46 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab14-h14 antibody amino acid sequence light chain <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 47 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab 14-h15 antibody amino acid sequence light chain <400> 47 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 48 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Humanized mab 14-h16 antibody amino acid sequence light chain <400> 48 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer p63a-SEQ <400> 49 cacaggtgtc cactcccagg t 21 <210> 50 <211> 481 <212> PRT <213> Artificial Sequence <220> <223> LB501 light chain sequence <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr Trp Ile Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile Tyr Pro Ser 165 170 175 Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe Lys Thr Arg Ala Thr Leu 180 185 190 Thr Val Asp Lys Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu 195 200 205 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Glu Gly Thr 210 215 220 Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp Phe Gly Tyr Trp Gly Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala 260 265 270 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 275 280 285 Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His Trp Tyr Gln 290 295 300 Gln Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr Leu Ala Ser Tyr 305 310 315 320 Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 325 330 335 Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro Glu Asp Phe Ala Val 340 345 350 Tyr Tyr Cys Gin His Ser Arg Asp Leu Pro Leu Thr Phe Gly Gly Gly 355 360 365 Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala Pro Ser Val Phe lie 370 375 380 Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val 385 390 395 400 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys 405 410 415 Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu 420 425 430 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 435 440 445 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 450 455 460 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 465 470 475 480 Cys <210> 51 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> LB501 heavy chain sequence (Pem heavy chain) <400> 51 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gin 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 52 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> LB502 light chain sequence (Pem light chain) <400> 52 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 145 150 155 160 Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 53 <211> 710 <212> PRT <213> Artificial Sequence <220> <223> LB502 heavy chain sequence <400> 53 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Val Gin 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr Trp Ile Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Gin Gly Leu Glu Trp lie Gly Asn lie Tyr Pro Ser 165 170 175 Ser lie Ser Thr Asn Tyr Asn Glu Lys Phe Lys Thr Arg Ala Thr Leu 180 185 190 Thr Val Asp Lys Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu 195 200 205 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Glu Gly Thr 210 215 220 Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp Phe Gly Tyr Trp Gly Gin 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Val Gin Ser Gly Val 260 265 270 Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 275 280 285 Gly Tyr Thr Phe Thr Asn Tyr Tyr Met Tyr Trp Val Arg Gin Ala Pro 290 295 300 Gly Gin Gly Leu Glu Trp Met Gly Gly lie Asn Pro Ser Asn Gly Gly 305 310 315 320 Thr Asn Phe Asn Glu Lys Phe Lys Asn Arg Val Thr Leu Thr Thr Asp 325 330 335 Ser Ser Thr Thr Thr Ala Tyr Met Glu Leu Lys Ser Leu Gln Phe Asp 340 345 350 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Asp Tyr Arg Phe Asp Met 355 360 365 Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 370 375 380 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 385 390 395 400 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 405 410 415 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 420 425 430 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 435 440 445 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr 450 455 460 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 465 470 475 480 Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 485 490 495 Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 500 505 510 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 515 520 525 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 530 535 540 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 545 550 555 560 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 565 570 575 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 580 585 590 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 595 600 605 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 610 615 620 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 625 630 635 640 Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 645 650 655 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 660 665 670 Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys 675 680 685 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 690 695 700 Ser Leu Ser Leu Gly Lys 705 710 <210> 54 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> LB503 light chain sequence <400> 54 Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro 35 40 45 Arg Leu Leu lie Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 145 150 155 160 Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Gin Val Thr His Gin Gin Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ala Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 225 230 235 240 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 245 250 255 Lys Ala Ser Gly Tyr Asn Phe Asn Ile Tyr Trp Ile Asn Trp Val Arg 260 265 270 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile Tyr Pro Ser 275 280 285 Ser Ile Ser Thr Asn Tyr Asn Glu Lys Phe Lys Thr Arg Ala Thr Leu 290 295 300 Thr Val Asp Lys Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu 305 310 315 320 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Glu Gly Thr 325 330 335 Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp Phe Gly Tyr Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 355 360 365 Gly Ser Gly Gly Gly Gly Ser Asp lie Gin Met Thr Gin Ser Pro Ser 370 375 380 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Arg Ala 385 390 395 400 Ser Gin Asp lie Arg Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Gly 405 410 415 Lys Ala Pro Lys Leu Leu lie Tyr Phe Thr Ser Thr Leu His Ser Gly 420 425 430 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 435 440 445 Thr lie Ser Ser Leu Gin Pro Gin Asp Phe Ala Thr Tyr Tyr Cys Gin 450 455 460 Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu 465 470 475 480 Ile Lys <210> 55 <211> 710 <212> PRT <213> Artificial Sequence <220> <223> LB504 heavy chain sequence <400> 55 Gln Val Gin Leu Val Gin Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gin Gin Asp Pro Glu 260 265 270 Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Gin Gin Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Gin Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gin Leu Pro Ser Ser Ile Gin Gin Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Ala Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val 450 455 460 Gln Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 465 470 475 480 Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Phe Asn He Tyr Trp He 485 490 495 Asn Trp Val Arg Gln Ala Pro Gly Gin Gly Leu Glu Trp He Gly Asn 500 505 510 Ile Tyr Pro Ser Ser He Ser Thr Asn Tyr Asn Glu Lys Phe Lys Thr 515 520 525 Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr Met Glu 530 535 540 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 545 550 555 560 Ser Glu Gly Thr Tyr Tyr Gly Gly Arg Tyr Glu Gly Asp Trp Phe Gly 565 570 575 Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 580 585 590 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He Gin Met Thr 595 600 605 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr He 610 615 620 Thr Cys Arg Ala Ser Gin Asp He Arg Asn Tyr Leu Asn Trp Tyr Gin 625 630 635 640 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu He Tyr Phe Thr Ser Thr 645 650 655 Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 660 665 670 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 675 680 685 Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly 690 695 700 Thr Lys Val Glu Ile Lys 705 710 <210> 56 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> LB506 light chain sequence (Nivo light chain) <400> 56 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 57 <211> 703 <212> PRT <213> Artificial Sequence <220> <223> LB506 heavy chain sequence <400> 57 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Cys 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala 450 455 460 Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 465 470 475 480 Gly Tyr Asn Phe Asn Ile Tyr Trp Ile Asn Trp Val Arg Gin Ala Pro 485 490 495 Gly Gin Gly Leu Glu Trp Ile Gly Asn Ile Tyr Pro Ser Ser Ile Ser 500 505 510 Thr Asn Tyr Asn Glu Lys Phe Lys Thr Arg Ala Thr Leu Thr Val Asp 515 520 525 Lys Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu 530 535 540 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Glu Gly Thr Tyr Tyr Gly 545 550 555 560 Gly Arg Tyr Glu Gly Asp Trp Phe Gly Tyr Trp Gly Gin Gly Thr Leu 565 570 575 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 580 585 590 Gly Gly Gly Ser Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser 595 600 605 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Asp 610 615 620 Ile Arg Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro 625 630 635 640 Lys Leu Leu Ile Tyr Phe Thr Ser Thr Leu His Ser Gly Val Pro Ser 645 650 655 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 660 665 670 Ser Leu Gin Pro Gin Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn 675 680 685 Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 690 695 700

Claims

1. An antibody or antigen-binding fragment thereof targeting Sirpa, characterized in that, The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, binds to human Sirpα-V1 and human Sirpα-V2, but weakly binds to or does not bind to human Sirpβ, Sirpγ, and does not bind to human T cells, and has the function of blocking the binding of Sirpα to CD47; The light chain variable region comprises CDRs of a VL CDR1 as shown in the amino acid sequence of SEQ ID NO: 11, a VL CDR2 as shown in the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 as shown in the amino acid sequence of SEQ ID NO: 13, and the heavy chain variable region comprises CDRs of a VH CDR1 as shown in the amino acid sequence of SEQ ID NO: 14, a VH CDR2 as shown in the amino acid sequence of SEQ ID NO: 15, and a VH CDR3 as shown in the amino acid sequence of SEQ ID NO: 16, according to the CCG numbering rule definition.

2. The antibody or antigen-binding fragment thereof targeted to Sirpa of claim 1, wherein, The light chain variable region is an amino acid sequence as shown in SEQ ID NO: 9 or a mutation thereof, and / or the heavy chain variable region is an amino acid sequence as shown in SEQ ID NO: 10 or a mutation thereof. The mutation is a deletion, substitution or insertion of one or more amino acid residues in the amino acid sequence of the light chain variable region and / or the heavy chain variable region, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the light chain variable region and / or the heavy chain variable region, and maintains or improves the binding of the antibody or antigen-binding fragment thereof to Sirpα.

3. The antibody or antigen-binding fragment thereof targeting Sirpa of claim 2, wherein, The light chain variable region is encoded by a nucleic acid as shown in SEQ ID NO: 7, and / or the heavy chain variable region is encoded by a nucleic acid as shown in SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof targeted to Sirpa of claim 2, wherein, The at least 85% sequence identity is at least 95% sequence identity.

5. The antibody or antigen-binding fragment thereof targeted to Sirpa of claim 2, wherein, The at least 85% sequence identity is at least 99% sequence identity.

6. The antibody or antigen-binding fragment thereof targeted to Sirpa of claim 2, wherein, The Sirpα-targeting antibody or antigen-binding fragment thereof further comprises a murine antibody constant region or a human antibody constant region; the murine antibody constant region includes a heavy chain constant region and a kappa or lambda type light chain constant region of murine IgG1, IgG2a, IgG2b3 or IgG3, and the human antibody constant region includes a heavy chain constant region and a kappa or lambda type light chain constant region of human IgG1, IgG2, IgG3 or IgG4.

7. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 6, wherein, When the Sirpα-targeting antibody or antigen-binding fragment thereof comprises a variable region of a murine antibody and a human antibody constant region, the human antibody constant region includes a heavy chain constant region and a kappa type light chain constant region of human IgG4, respectively as shown in the amino acid sequences of SEQ ID NO: 28 and SEQ ID NO:

27.

8. The antibody or antigen-binding fragment thereof targeted to Sirpa of claim 1, wherein, The Sirpα-targeting antibody is a humanized antibody. The framework region of the humanized antibody includes a human antibody heavy chain framework region and a human antibody light chain framework region.

9. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 8, wherein, The light chain variable region of the humanized antibody comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-34 or a mutation thereof; and / or, the heavy chain variable region sequence of the humanized antibody comprises an amino acid sequence as shown in any one of SEQ ID NOs: 35-41 or a mutation thereof; the mutation is a deletion, substitution or insertion of one or more amino acid residues in the amino acid sequence of the light chain variable region and / or heavy chain variable region, and the mutated amino acid sequence has at least 85% sequence identity to the amino acid sequence of the light chain variable region and / or heavy chain variable region, and maintains or improves the binding of the antibody or antigen-binding fragment thereof to Sirpa.

10. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 9, wherein, The at least 85% sequence identity is at least 90% sequence identity.

11. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 9, wherein, The at least 85% sequence identity is at least 95% sequence identity.

12. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 9, wherein, The at least 85% sequence identity is at least 99% sequence identity.

13. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 9, wherein, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 35; or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 30, and the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 36-41; or, the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 31-34, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36; or, the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29 or 31-34, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:

39.

14. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 8, wherein, The light chain of the antibody or antigen-binding fragment thereof comprises a human antibody kappa or lambda type light chain constant region or a mutation thereof; and / or, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or a mutation thereof.

15. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 14, wherein, The light chain of the antibody or antigen-binding fragment thereof comprises a human antibody kappa type light chain constant region; and / or, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4.

16. The antibody or antigen-binding fragment thereof targeted to Sirpα of claim 14, wherein, The light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48 or a mutation thereof; and / or, the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 43 or a mutation thereof.

17. The antibody or antigen-binding fragment thereof targeting Sirpa of claim 14, comprising a light chain and a heavy chain: the light chain is as shown in any one of amino acid sequences of SEQ ID NOs: 42 or 44-48, and the heavy chain is as shown in the amino acid sequence of SEQ ID NO:

43.

18. The Sirpα- targeting antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the Sirpα- targeting antibody or antigen-binding fragment thereof comprises an immunoglobulin, a Fab, a Fab', a F(ab')2, a Fv, or a scFv.

19. A bispecific antibody comprising a first protein domain and a second protein domain, characterized in that, the first protein functional region is the Sirpα- targeting antibody or antigen-binding fragment thereof of any one of claims 1-18; and the second protein functional region is an antibody or antigen-binding fragment thereof that targets a non-Sirpα antigen; the second protein functional region is Nivolumab or Pembrolizumab; the second protein functional region is an immunoglobulin, and the first protein functional region is two scFvs; wherein the scFv comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are connected by a linker; the scFv is connected to the immunoglobulin by a linker; when the second protein functional region is Pembrolizumab, the immunoglobulin comprises the amino acid sequences of the light chain variable region, the light chain constant region as a kappa chain, the heavy chain variable region, and the heavy chain constant region of hlgG4 of Pembrolizumab; the light chain variable region of the scFv is the light chain variable region of the amino acid sequence as set forth in SEQ ID NO: 29, and the heavy chain variable region of the scFv is the heavy chain variable region of the amino acid sequence as set forth in SEQ ID NO: 39; the structure of the scFv is light chain variable region-linker-heavy chain variable region, the N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region is respectively connected to the C-terminus or N-terminus of the light chain or heavy chain of the immunoglobulin by a linker; or the structure of the scFv is heavy chain variable region-linker-light chain variable region, the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region is respectively connected to the C-terminus or N-terminus of the light chain or heavy chain of the immunoglobulin by a linker; the two scFvs are symmetrically connected to the C-terminus or N-terminus of the light chain or heavy chain of the immunoglobulin; or, when the second protein functional region is Nivolumab, the immunoglobulin comprises the amino acid sequences of the light chain variable region, the light chain constant region as a kappa chain, the heavy chain variable region, and the heavy chain constant region of hlgG4 of Nivolumab; the structure of the scFv is heavy chain variable region-linker-light chain variable region; the N-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the C-terminus of the two heavy chains of the immunoglobulin by a linker; and the light chain variable region of the scFv is the light chain variable region of the amino acid sequence as set forth in SEQ ID NO: 29, and the heavy chain variable region of the scFv is the heavy chain variable region of the amino acid sequence as set forth in SEQ ID NO:

39.

20. The bispecific antibody of claim 19, wherein, the linker is (G4S) w ; w is an integer between 0 and 10.

21. The bispecific antibody of claim 20, wherein the linker is (G4S) w , and w is 1, 2, 3, or 4.

22. The bispecific antibody of claim 19, wherein the first protein functional region is an antibody or antigen-binding fragment thereof that targets Sirpα, and the second protein functional region is an antibody or antigen-binding fragment thereof that targets a non-Sirpα antigen. When the second protein functional region is Pembrolizumab, the C-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the N-terminus of the two heavy chains of the immunoglobulin through a linker; and the light chain variable region of the scFv is a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 39; or the C-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the N-terminus of the two light chain variable regions of the immunoglobulin through a linker; and the light chain variable region of the scFv is a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 39; or the N-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the C-terminus of the two heavy chains of the immunoglobulin through a linker; and the light chain variable region of the scFv is a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 39; or the N-terminus of the heavy chain variable region of the two scFvs is symmetrically connected to the C-terminus of the two light chains of the immunoglobulin through a linker; and the light chain variable region of the scFv is a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:

39.

23. The bispecific antibody of any one of claims 19-22, wherein, The bispecific antibody comprises the following light chain amino acid sequence and heavy chain amino acid sequence: a light chain amino acid sequence as shown in SEQ ID NO: 50 and a heavy chain amino acid sequence as shown in SEQ ID NO: 51; or a light chain amino acid sequence as shown in SEQ ID NO: 52 and a heavy chain amino acid sequence as shown in SEQ ID NO: 53; or a light chain amino acid sequence as shown in SEQ ID NO: 54 and a heavy chain amino acid sequence as shown in SEQ ID NO: 51; or a light chain amino acid sequence as shown in SEQ ID NO: 52 and a heavy chain amino acid sequence as shown in SEQ ID NO: 55; or a light chain amino acid sequence as shown in SEQ ID NO: 56 and a heavy chain amino acid sequence as shown in SEQ ID NO:

57.

24. An isolated nucleic acid encoding the Sirpα-targeting antibody or antigen binding fragment thereof of any one of claims 1-18 or the bispecific antibody of any one of claims 19-23.

25. A recombinant expression vector comprising the isolated nucleic acid of claim 24.

26. The recombinant expression vector of claim 25, wherein, The expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector. The expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

27. A transformant comprising the recombinant expression vector of claim 25 or 26 in a host cell.

28. The transformant of claim 27, wherein The host cell is a prokaryotic cell and / or a eukaryotic cell.

29. The transformant of claim 28, wherein, The prokaryotic cell is an E. coli cell, and the eukaryotic cell is a HEK293 cell or a CHO cell.

30. The transformant of claim 29, wherein The E. coli cell is TG1 or BL21.

31. A method for producing an antibody or antigen-binding fragment thereof targeting Sirpa, comprising culturing the transformant of any one of claims 27-30, and obtaining the antibody or antigen-binding fragment thereof targeting Sirpa from the culture.

32. A pharmaceutical composition comprising, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof targeting Sirpa of any one of claims 1-18 and / or the bispecific antibody of any one of claims 19-23, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein, The pharmaceutical composition further comprises another anti-tumor antibody as an active ingredient.

34. Use of the antibody or antigen-binding fragment thereof targeting Sirpa of any one of claims 1-18, the bispecific antibody of any one of claims 19-23, or the pharmaceutical composition of claim 32 or 33 in the manufacture of a medicament for treating a tumor.

Images