Anti-CD40 antibody and its uses

Abstract

The present invention relates to an anti-CD40 antibody or antigen-binding fragment thereof that specifically recognizes and weakly stimulates human, cynomolgus monkey and mouse CD40 molecules, as well as immunoconjugates, pharmaceutical compositions and combination products comprising said anti-CD40 antibody or antigen-binding fragment thereof.The present invention further relates to a nucleic acid encoding said anti-CD40 antibody or antigen-binding fragment thereof, and a host cell comprising said nucleic acid, as well as a method for producing said anti-CD40 antibody or antigen-binding fragment thereof.The present invention further relates to the use of said anti-CD40 antibody or antigen-binding fragment thereof in the prevention or treatment of a tumor or an infectious disease in a subject.

Description

Technical Field

[0001] The present invention relates to an anti-CD40 antibody or an antigen-binding fragment thereof. Specifically, the present invention relates to an anti-CD40 antibody or an antigen-binding fragment thereof that specifically recognizes human, cynomolgus monkey, and mouse CD40 molecules and weakly stimulates the CD40 molecules, and to an immunoconjugate, a pharmaceutical composition, and a combination product containing the anti-CD40 antibody or an antigen-binding fragment thereof. The present invention further relates to a nucleic acid encoding the anti-CD40 antibody or an antigen-binding fragment thereof, a host cell containing the nucleic acid, and a method for producing the anti-CD40 antibody or an antigen-binding fragment thereof. The present invention further relates to the use of the anti-CD40 antibody or an antigen-binding fragment thereof in the prevention or treatment of tumors or infectious diseases in a subject.

Background Art

[0002] CD40 agonists (such as selicrelumab, dacetuzumab, APX005M, etc.) have been shown by clinical trials to exhibit clinical activity in various indications (Hassan SB et al., Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials. Immunopharmacol Immunotoxicol 2014;36:96-104). However, these studies have also shown that the CD40 agonists are associated with adverse events that result in dose-limiting toxicity. The most common adverse events include cytokine release syndrome (CRS) and hepatotoxicity. The CD40 agonists generally used in current clinical trials are those with normal Fc function, and such agonists can be classified into two types according to agonist activity. Here, some agonists can strongly activate the immune response nonspecifically and excessively, so they are highly toxic and not very suitable as therapeutic agents. However, some other agonists require a crosslinking effect to form anti-CD40 monoclonal antibodies with immune activity, and by activating immune cells expressing CD40 in a limited and controllable manner to form a local immune enhancement response, they kill tumor cells within a limited range.

[0003] In recent years, the therapeutic index (TI) has become an important indicator for evaluating the safety of drugs. The TI compares the amount of drug that causes a therapeutic effect with the amount of drug that causes toxicity, meaning that the higher the TI and the greater the safety, the higher the drug efficacy and the lower the toxicity at the dose of the drug administered to patients. In the prior art, currently known anti-CD40 antibodies have a low therapeutic index, and at the dose actually administered to patients, the ratio of the immunocyte killing activity formed by the antibody at the lesion site to the non-specific agonist activity (e.g., hepatotoxicity) caused at other sites is not high. Therefore, in this field, there is a need to develop a new CD40 agonist that can provide sufficient immune stimulation and reduce adverse events caused by conventional CD40 agonists.

[0004] In addition, in the development of a new CD40 agonist, preclinical animal model evaluation is an important part of evaluating the safety and therapeutic effect of the drug. Rodents (e.g., mice) are widely used in the establishment of animal models of human diseases. However, anti-CD40 antibodies that do not cross-react with mouse CD40 (e.g., selicrelumab developed by Roche) need to be used in human CD40 transgenic mouse tumor models to reflect their in vivo efficacy and safety. In the efficacy test of anti-CD40 bispecific antibodies in mouse models, there is also an urgent need to develop agonist anti-CD40 antibodies that simultaneously have mouse CD40 cross-reactivity.

Summary of the Invention

[0005] The present invention provides an agonist anti-CD40 antibody with a high therapeutic index, which can provide sufficient immune stimulation in a subject and attenuate the adverse effects of non-specific immune activation caused by CD40 activation, which is (1) Bind to and activate CD40 with high affinity as measured by the ForteBio kinetic binding assay, e.g., bind to CD40, such as human CD40, cynomolgus monkey CD40, and mouse CD40, at about 10 -7 M to about 10 -10 M, (2)Enhancing the binding of CD40 and CD40L, (3)Activating antigen-presenting cells by binding to CD40 expressed on antigen-presenting cells, for example, the antigen-presenting cells include dendritic cells (DCs), B cells, monocytes, and macrophages, (4)Inducing CD95 expression by B cells expressing CD40, (5)Significantly enhancing the B cell-mediated immune response when a cross-linking effect is formed, (6)Having one or more of the characteristics of hardly enhancing or weakly enhancing the B cell-mediated immune response when no cross-linking effect is formed.

[0006] The anti-CD40 antibody of the present invention has a complete human sequence and is a human antibody. The anti-CD40 antibody of the present invention has minimal immunogenicity against human subjects and induces fewer anti-drug antibodies (ADA), thereby minimizing the clearance rate of the therapeutic anti-CD40 antibody related to ADA, and it is expected that human subjects have good tolerance to the therapeutic anti-CD40 antibody.

[0007] In some embodiments, the anti-CD40 antibody of the present invention includes a light chain variable region and a heavy chain variable region. The light chain variable region includes three complementarity-determining regions, namely LCDR1, LCDR2, and LCDR3 respectively. The heavy chain variable region includes three complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3 respectively. Here, (a) LCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, or a variant with two or fewer or one or fewer amino acid changes of SEQ ID NO: 1, (b) LCDR2 includes any one of the amino acid sequences selected from SEQ ID NO: 2, 5, 6, 7, or a variant with two or fewer or one or fewer amino acid changes of any one of the amino acid sequences of SEQ ID NO: 2, 5, 6, 7, (c) LCDR3 comprises any one amino acid sequence selected from SEQ ID NO: 3 and 4, or a variant with two or fewer or one or fewer amino acid changes in any one amino acid sequence of SEQ ID NO: 3 and 4, (d) HCDR1 comprises any one amino acid sequence selected from SEQ ID NO: 8, 12, 14, and 20, or a variant with two or fewer or one or fewer amino acid changes in any one amino acid sequence of SEQ ID NO: 8, 12, 14, and 20, (e) HCDR2 comprises any one amino acid sequence selected from SEQ ID NO: 9 and 21, or a variant with two or fewer or one or fewer amino acid changes in any one amino acid sequence of SEQ ID NO: 9 and 21, (f) HCDR3 comprises any one amino acid sequence selected from SEQ ID NO: 10, 11, 13, 15, 16, 17, 18, and 19, or a variant with two or fewer or one or fewer amino acid changes in any one amino acid sequence of SEQ ID NO: 10, 11, 13, 15, 16, 17, 18, and 19, Here, the amino acid change is an addition, deletion, or substitution of an amino acid.

[0008] In some embodiments, the anti-CD40 antibody of the present invention is (i) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 or one or two or fewer amino acid changes of SEQ ID NO: 10, (ii) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 4 or one or two or fewer amino acid changes of SEQ ID NO: 4, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 or one or two or fewer amino acid changes of SEQ ID NO: 10, (iii) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 11 or one or two or fewer amino acid changes of SEQ ID NO: 11 (iv) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 5 or one or two or fewer amino acid changes of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 12 or one or two or fewer amino acid changes of SEQ ID NO: 12, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 or one or two or fewer amino acid changes of SEQ ID NO: 10 (v) An LCDR1 containing the amino acid sequence SEQ ID NO: 1 or containing one or two or less amino acid changes of SEQ ID NO: 1, an LCDR2 containing the amino acid sequence SEQ ID NO: 2 or containing one or two or less amino acid changes of SEQ ID NO: 2, and an LCDR3 containing the amino acid sequence SEQ ID NO:3 or containing one or two or less amino acid changes of SEQ ID NO: 3, as well as an HCDR1 containing the amino acid sequence SEQ ID NO: 8 or containing one or two or less amino acid changes of SEQ ID NO: 8, an HCDR2 containing the amino acid sequence SEQ ID NO: 9 or containing one or two or less amino acid changes of SEQ ID NO: 9, and an LCDR3 containing the amino acid sequence SEQ ID NO:13 or containing one or two or less amino acid changes of SEQ ID NO: 13 (vi) An LCDR1 containing the amino acid sequence SEQ ID NO: 1 or containing one or two or less amino acid changes of SEQ ID NO: 1, an LCDR2 containing the amino acid sequence SEQ ID NO: 6 or containing one or two or less amino acid changes of SEQ ID NO: 6, and an LCDR3 containing the amino acid sequence SEQ ID NO:3 or containing one or two or less amino acid changes of SEQ ID NO: 3, as well as an HCDR1 containing the amino acid sequence SEQ ID NO: 14 or containing one or two or less amino acid changes of SEQ ID NO: 14, an HCDR2 containing the amino acid sequence SEQ ID NO: 9 or containing one or two or less amino acid changes of SEQ ID NO: 9, and an LCDR3 containing the amino acid sequence SEQ ID NO:10 or containing one or two or less amino acid changes of SEQ ID NO: 10 (vii) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 15 or one or two or fewer amino acid changes of SEQ ID NO: 15 (viii) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 16 or one or two or fewer amino acid changes of SEQ ID NO: 16 (ix) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 17 or one or two or fewer amino acid changes of SEQ ID NO: 17 (x) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or fewer amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or fewer amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or fewer amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or fewer amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or fewer amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 18 or one or two or fewer amino acid changes of SEQ ID NO: 18 (xi) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or less amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2 or one or two or less amino acid changes of SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or less amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8 or one or two or less amino acid changes of SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9 or one or two or less amino acid changes of SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 19 or one or two or less amino acid changes of SEQ ID NO: 19, or, (xii) LCDR1 containing the amino acid sequence SEQ ID NO: 1 or one or two or less amino acid changes of SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 7 or one or two or less amino acid changes of SEQ ID NO: 7, and LCDR3 containing the amino acid sequence SEQ ID NO: 3 or one or two or less amino acid changes of SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 20 or one or two or less amino acid changes of SEQ ID NO: 20, HCDR2 containing the amino acid sequence SEQ ID NO: 21 or one or two or less amino acid changes of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 or one or two or less amino acid changes of SEQ ID NO: 10.

[0009] In some embodiments, the anti - CD40 antibody of the present invention is LCDR1 containing something like the amino acid sequence RSSQGIRSSLA (SEQ ID NO: 1), amino acid sequence GX 1 SSLX 2 X 3LCDR2 containing something like (SEQ ID NO: 56), and the amino acid sequence QQLX 4 LCDR3 containing something like SFPST (SEQ ID NO: 57), wherein X 1 is A or G, and X 2 is E or L, and X 3 is G or V, and X 4 is N or A, and the amino acid sequence GFTX 5 GSYEMX 6 HCDR1 containing something like (SEQ ID NO: 58), the amino acid sequence YISSX 7 HCDR2 containing something like GETTD (SEQ ID NO: 59), and the amino acid sequence DVFFFX 8 X 9 SX 10 X 11 X 12 X 13 HCDR3 containing something like AYGMDV (SEQ ID NO: 60), wherein X 5 is F, A or P, and X 6 is N or D, and X 7 is S or A, and X 8 is D or S, and X 9 is S or P, and X 10 is G or R, and X 11 is D, P, S or F, and X 12 is P, N or R, and X 13 is G or H, including such things.

[0010] In some specific embodiments, the anti - CD40 antibody of the present invention is (i) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10, (ii) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 4, as well as HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 (iii) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, as well as HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 11 (iv) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 5, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, as well as HCDR1 containing the amino acid sequence SEQ ID NO: 12, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 (v) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, as well as HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 13 (vi) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 6, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, as well as HCDR1 containing the amino acid sequence SEQ ID NO: 14, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 10 (vii) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 15, (viii) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 16, (ix) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 17, (x) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 18, (xi) LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 2, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, and HCDR1 containing the amino acid sequence SEQ ID NO: 8, HCDR2 containing the amino acid sequence SEQ ID NO: 9, and LCDR3 containing the amino acid sequence SEQ ID NO: 19, or, (xii) It includes LCDR1 containing the amino acid sequence SEQ ID NO: 1, LCDR2 containing the amino acid sequence SEQ ID NO: 7, and LCDR3 containing the amino acid sequence SEQ ID NO: 3, as well as HCDR1 containing the amino acid sequence SEQ ID NO: 20, HCDR2 containing the amino acid sequence SEQ ID NO: 21, and LCDR3 containing the amino acid sequence SEQ ID NO: 10.

[0011] In some embodiments, the anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein (i) the light chain variable region contains any one of the amino acid sequences of SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains any one of the amino acid sequences of SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55, or (ii) the light chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of the amino acid sequences of SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of the amino acid sequences of SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55.

[0012] In some embodiments, the anti-CD40 antibody of the present invention is (i) a light chain variable region containing the amino acid sequence SEQ ID NO: 23 and a heavy chain variable region containing the amino acid sequence SEQ ID NO: 25, (ii) a light chain variable region containing the amino acid sequence SEQ ID NO: 29 and a heavy chain variable region containing the amino acid sequence SEQ ID NO: 25, (iii) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:31, (iv) A light chain variable region containing the amino acid sequence SEQ ID NO:33, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:35, (v) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:37, (vi) A light chain variable region containing the amino acid sequence SEQ ID NO:39, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:41, (vii) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:43, (viii) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:45, (ix) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:47, (x) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:49, (xi) A light chain variable region containing the amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:51, or, (xii) Comprising a light chain variable region containing the amino acid sequence SEQ ID NO:53, and a heavy chain variable region containing the amino acid sequence SEQ ID NO:55, Preferably, the anti-CD40 antibody or antigen-binding fragment is a fully human antibody.

[0013] In some embodiments, the anti-CD40 antibody of the present invention is an IgG1, IgG2, IgG3 or IgG4 antibody, preferably it is an IgG1 or IgG4 antibody, and more preferably it is a human IgG1 or human IgG4 antibody.

[0014] In some embodiments, the anti-CD40 antibody of the present invention is an antigen-binding fragment, including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab, diabody.

[0015] In some embodiments, the anti-CD40 antibody of the present invention has the glycosylation site removed in the CH2 domain of its immunoglobulin Fc region. For example, the N297 residue in the CH2 domain of the human IgG Fc region is mutated to remove the glycosylation site, for example, the N297 residue is changed to Gly, Ala, Gln, Asp or Glu, preferably, the N297 residue is changed to Gln.

[0016] In a second aspect, the present invention provides a method for producing the anti-CD40 antibody of the present invention, the method comprising culturing a host cell into which a nucleic acid encoding the anti-CD40 antibody of the present invention or an expression vector containing the nucleic acid is introduced under conditions suitable for the expression of the nucleic acid encoding the anti-CD40 antibody of the present invention, and isolating the anti-CD40 antibody, and optionally, the method further comprises recovering the anti-CD40 antibody from the host cell.

[0017] In a third aspect, the present invention provides an immunoconjugate comprising the anti-CD40 antibody of the present invention and other substances, such as cytotoxic agents.

[0018] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the anti-CD40 antibody or immunoconjugate of the present invention, and an optional pharmaceutical adjuvant.

[0019] In some embodiments, the present invention provides a pharmaceutical composition comprising the anti-CD40 antibody or immunoconjugate of the present invention, and other therapeutic agents, and an optional pharmaceutical adjuvant, preferably, the other therapeutic agents are selected from chemotherapeutic agents, other antibodies (such as anti-PD-1 antibody or anti-PD-L1 antibody), cytotoxic agents.

[0020] In some embodiments, the present invention provides a combination product comprising an anti-CD40 antibody or immunoconjugate of the present invention and one or more other therapeutic agents, such as chemotherapeutic agents, cytotoxic agents, other antibodies, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0021] In a fifth aspect, the present invention provides a method for preventing or treating a tumor or infectious disease of a subject or individual in a subject, comprising administering to the subject an effective amount of an anti-CD40 antibody, immunoconjugate, pharmaceutical composition, or combination product of the present invention.

[0022] In some embodiments, the tumors prevented or treated by the anti-CD40 antibody, immunoconjugate, pharmaceutical composition, or combination product of the present invention are cancers, such as cancers that express the CD40 molecule or cancers that do not express the CD40 molecule, or the infectious diseases prevented or treated by the anti-CD40 antibody, immunoconjugate, pharmaceutical composition, or combination product of the present invention are, for example, bacterial infections, viral infections, fungal infections, or protozoal infections, preferably, the infectious disease is a chronic infection, and the immunity of the subject or individual is reduced in the infection.

[0023] In a sixth aspect, the present invention provides a kit for CD40 in a test sample, the kit comprising an anti-CD40 antibody of the present invention, (a) for performing the step of contacting the sample with the anti-CD40 antibody of the present invention, and (b) for performing the step of detecting the formation of a complex between the anti-CD40 antibody and CD40, and optionally, the anti-CD40 antibody is detectably labeled, thereby determining the CD40 expression level in a sample from a subject or individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of explaining the present invention, the current preferred embodiments are shown in the figures. However, it should be understood that the present invention is not limited to the exact arrangements and means of the embodiments shown in the figures.

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Modes for Carrying Out the Invention

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from the specification, drawings, and appended claims.

[0026] I. Definitions

[0027] For the purposes of this specification, the following definitions are used, and where appropriate, terms used in the singular also include the plural and vice versa. It is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0028] The term "about", when used in conjunction with a number or numerical value, means a number or numerical value within a range from 5% less than the specified number or numerical value to 5% greater than the specified number or numerical value.

[0029] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0030] As used herein, the terms "comprising" or "including" cover cases where the stated elements, integers, or steps are present, unless otherwise specified. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to cover an antibody variable region consisting of that specific sequence.

[0031] The term "CD40-expressing cell" refers to any cell that expresses the CD40 molecule, including, but not limited to, antigen-presenting cells (APCs) such as dendritic cells (DCs), B cells, macrophages, and monocytes. CD40 is also expressed on other types of cells, such as epithelial cells, endothelial cells, and platelets. CD40 expression has been confirmed on many tumor cells, including B cell lymphoma and renal carcinoma cells. In one specific embodiment, CD40-expressing cells include cell lines that express CD40, such as Jurkat cells, Raji cells, Ramos cells, and Daudi cells. In another embodiment, the CD40-expressing cells are tumor cells or cancer cells. In another embodiment, the CD40-expressing cells include B cells, NK cells, and T cells that are found to infiltrate tumors, also known as tumor-infiltrating lymphocytes.

[0032] The term "antigen-presenting cell" or "APC" refers to a cell that displays an exogenous antigen complexed with MHC on its surface. T cells recognize the complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblastoid cells, and dendritic cells (DCs) derived from monocytes.

[0033] The term "antigen presentation" refers to the process by which an APC captures an antigen and enables its recognition by T cells, for example, by processing the antigen into components of MHC-I and / or MHC-II conjugates.

[0034] "MHC molecules" include two types of molecules, MHC class I and MHC class II. MHC class I molecules present antigens to specific CD8+ T cells, and MHC class II molecules present antigens to specific CD4+ T cells. Antigens delivered exogenously to APCs are mainly for binding to MHC class II. Conversely, antigens delivered endogenously to APCs are mainly for binding to MHC class I.

[0035] The term "T cell-mediated response" refers to any response mediated by T cells (including effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells)). T cell-mediated responses include, for example, cytotoxicity and proliferation of T cells.

[0036] The term "antibody" is used herein in its broadest sense and refers to proteins containing an antigen-binding site, natural and artificial antibodies covering various structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments, but not limited thereto.

[0037] "Antibody fragment" or "antigen-binding fragment" refers to a molecule different from a whole antibody that includes a part of a whole antibody and binds to an antigen to which the whole antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody (e.g., scFv), single-domain antibody, bivalent or bispecific antibody or fragments thereof, camelid antibody (heavy-chain antibody), and bispecific or multispecific antibody formed by antibody fragments.

[0038] "Cross-reactivity" refers to the ability of the antibodies of the present invention to bind to CD40 from different species. For example, an antibody of the present invention that binds to human CD40 can also bind to CD40 of another species. Cross-reactivity is measured by detecting specific reactivity with purified CD40 antigen in a binding assay (e.g., SPR, ELISA), or by binding to or otherwise functionally interacting with physiologically CD40-expressing cells. Methods for determining cross-reactivity include, for example, Octet TM Biolayer interferometry (BLI) using a QKe instrument or Biacore using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) TM Surface plasmon resonance (SPR) analysis or flow cytometry techniques.

[0039] "Complementary determining region" or "CDR region" or "CDR" is a region in the antibody variable domain where loops with highly variable sequences and defined structures ("hypervariable loops") are formed and / or which contains antigen - contacting residues ("antigen - contact points"). CDRs are mainly responsible for binding to antigen epitopes. The CDRs of the heavy chain are usually called CDR1, CDR2 and CDR3 and are numbered in order from the N - terminus. In a given heavy - chain variable - region amino - acid sequence, the exact amino - acid sequence boundaries of each CDR can be determined by any one or a combination of several known antibody CDR assignment systems, said assignment systems including, for example, Chothia (Chothia et al. (1989) Nature 342: 877 - 883, Al - Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927 - 948 (1997)) based on the three - dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a number of crystal structures.

[0040] Unless otherwise specified, in the present invention, the terms "CDR" or "CDR sequence" cover CDR sequences determined in any of the above - mentioned ways.

[0041] The CDR may also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any one of the CDRs exemplified in the present invention). In one embodiment, the CDRs of the antibodies of the present invention have their positions determined based on the AbM numbering protocol.

[0042] Unless otherwise specified, in the present invention, when referring to residue positions in the antibody variable region and CDRs (including heavy chain variable region residues), it refers to the numbering positions based on the AbM numbering system.

[0043] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although the CDRs differ between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. By determining the minimally overlapping region using at least two of the Kabat, Chothia, AbM, and Contact methods, a "minimal binding unit" for antigen binding can be provided. The minimal binding unit may be a subpart of the CDR. As will be apparent to those skilled in the art, due to the structure of the antibody and protein folding, residues in other parts of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR presented herein. For example, in a variant of one CDR, the amino acid residues of the minimal binding unit may remain as they are, but other CDR residues defined based on Kabat or Chothia or AbM may be substituted with conserved amino acid residues.

[0044] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source and utilizing a human antibody library or other human antibody coding sequence. The definition of a human antibody clearly excludes humanized antibodies containing non-human antigen-binding residues.

[0045] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, and said region includes at least a portion of the constant region. The term includes both native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl group terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991, unless otherwise specified.

[0046] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody generally have similar structures, where each domain includes four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co. page 91 (2007)). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0047] As used herein, the terms "bind" or "specifically bind" mean that the binding interaction is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA), SPR or biolayer interferometry techniques or other common binding assays known in the art.

[0048] The term "costimulatory molecule" refers to cell surface molecules and their ligands that provide costimulatory signals for the full activation of T cells or B cells. Costimulatory molecules refer to cell surface molecules other than antigen receptors or their ligands that are useful for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.

[0049] The term "cytokine" is a general term for proteins released by one cell population and acting on other cells as an intercellular medium. Examples of such cytokines include lymphokines such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, monokines, interleukins (IL), tumor necrosis factors such as TNF-α or TNF-β, and other polypeptide factors including γ-interferon.

[0050] An "immunoconjugate" refers to an antibody conjugated to one or more other substances (including, but not limited to, cytotoxic agents or labels).

[0051] The terms "induce" and "increase" as used in CDC or ADCC induction mean to stimulate a specific direct cell killing mechanism. For example, in one embodiment, the anti-CD40 antibody of the present invention results in the lysis of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of tumor cells or cells infected with viruses, bacteria, etc. by ADCC through the activation of CD40-expressing cells.

[0052] The term "agonist" refers to increasing some parameters (e.g., activity) of a given molecule (e.g., a costimulatory molecule). For example, this term includes substances that improve the activity of a given molecule (e.g., CD40) by at least 5%, 10%, 20%, 30%, 40% or more. Therefore, agonist action is not necessarily 100%.

[0053] The "functional Fc region" has the "effector function" of the native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor, BCR), etc. Such effector functions generally require the Fc region to associate with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays, such as those disclosed herein.

[0054] "Effector function" refers to biological activities that result from the antibody Fc region and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0055] The term "Fc receptor" refers to an Fc receptor that, after binding to the immunoglobulin Fc region, elicits a signal transduction event that stimulates the cell bearing the receptor to perform an effector function. Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0056] The term "effective amount" refers to the amount or dosage of the anti-CD40 antibody or conjugate or composition of the present invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prophylaxis. The effective amount can be readily determined by an attending physician, who is skilled in the art, taking into account various factors such as the mammalian species, body weight, age and general health status, the specific disease involved, the degree or severity of the disease, the response of the individual patient, the specific antibody being administered, the mode of administration, the bioavailability characteristics of the formulation being administered, the dosing regimen selected, and the use of any concomitant therapies.

[0057] "Therapeutically effective amount" refers to the amount that effectively achieves the desired therapeutic result at the required dosage and over the required period. The therapeutically effective amount of an antibody or antibody fragment or their conjugate or composition can vary depending on various factors such as the disease state, the age, sex and body weight of the individual, and the ability of the antibody or antibody moiety to elicit the desired response in the individual. The therapeutically effective amount is also the amount at which any toxic or adverse effects of the antibody or antibody fragment or their conjugate or composition do not outweigh the therapeutic beneficial effects. For an untreated subject, a "therapeutically effective amount" preferably suppresses a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70%, still more preferably by at least about 80% or 90%. The ability of a compound to suppress a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

[0058] "Prophylactically effective amount" refers to the amount that effectively achieves the desired prophylactic result at the required dosage and over the required period. Usually, since the prophylactic dosage is used in a subject before or at an earlier stage of the disease than the later stage of the disease, the prophylactically effective amount is smaller than the therapeutically effective amount.

[0059] "Therapeutic index" (TI) usually refers to the ratio of the median lethal dose (LD50) to the median effective dose (ED50) and is an index of the safety of a drug.

[0060] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, farm animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., non-human primates such as humans and monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0061] The terms "tumor" and "cancer" are used interchangeably herein and cover solid tumors and liquid tumors.

[0062] The terms "cancer" and "cancerous" refer to a physiological disease in mammals in which cell growth cannot be controlled.

[0063] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", and "tumor" are not mutually exclusive when referred to herein.

[0064] "Tumor immune escape" refers to the process by which a tumor escapes immune recognition and elimination. Thus, as a treatment concept, tumor immunity is "treated" when such escape is weakened and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination.

[0065] The term "infectious disease" refers to a disease caused by a pathogen and includes, for example, viral infection, bacterial infection, fungal infection, or protozoan, for example, parasitic infection.

[0066] The term "chronic infection" refers to an infection in which an infectious agent (e.g., a pathogen such as a virus, bacterium, protozoan such as a parasite, fungus, etc.) has induced an immune response in the infected host but has not been removed or eliminated from the host as in the case of acute infection. Chronic infections can be persistent, latent, or slow.

[0067] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label), or in the case of an enzymatic label, it can catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reacting it with another reagent that is directly labeled. Examples of indirect labeling include the detection of a first antibody using a fluorescently labeled second antibody, and the end-labeling of a DNA probe having biotin so that it can be detected with fluorescently labeled streptavidin.

[0068] An "isolated" anti-CD40 antibody refers to one that has been isolated from the components of its natural environment. In some embodiments, the anti-CD40 antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For an overview of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0069] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes nucleic acid molecules that are contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An "isolated nucleic acid encoding an anti-CD40 antibody" refers to one or more nucleic acid molecules that encode a chain or a fragment of an anti-CD40 antibody, including such nucleic acid molecules in a single vector or individual vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0070] The calculation of array identity between arrays is performed as follows.

[0071] To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison). In one preferred embodiment, for comparison, the length of the reference sequence to be aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. Thereafter, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecule is the same at this position.

[0072] Array comparison and calculation of the identity ratio between two arrays can be realized by using mathematical algorithms. In one preferred embodiment, Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithms incorporated into the GAP program of the GCG software package (available from http: / / www.gcg.com), the Blossum 62 matrix or the PAM250 matrix, and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 are used to determine the identity ratio between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available from http: / / www.gcg.com), the NWSgapdna.CMP matrix, and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6 are used to determine the identity ratio between two nucleotide sequences. A particularly preferred parameter set (and the parameter set to be used unless otherwise stated) is the Blossum 62 score matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0073] Furthermore, the identity ratio between two amino acid sequences or nucleotide sequences can be determined by using the E. Meyers and W. Miller algorithms incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17) with the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4).

[0074] Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against common databases to identify, for example, other family member sequences or related sequences.

[0075] The term "pharmaceutical composition" refers to a composition that exists in a form that permits the biological activity of the active ingredient(s) contained therein to be effective and that contains no other ingredients that have unacceptable toxicity for the subject to whom the composition is administered.

[0076] The term "pharmaceutical adjuvant" refers to a diluent, adjuvant, vector, excipient, stabilizer, etc. that is administered together with an active substance.

[0077] As used herein, "treatment" refers to slowing, interrupting, halting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, condition, state, or disease. Desired therapeutic effects include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, reduction in the rate of disease progression, improvement or alleviation of a disease state, and remission or improvement of the prognosis. In some embodiments, the antibody molecules of the invention are used to delay or slow the progression of a disease.

[0078] As used herein, "prevention" includes suppression of the occurrence or progression of a disease or condition or the symptoms of a particular disease or condition. In some embodiments, a subject having a family history of cancer is a candidate for a preventive regimen. Typically, in the context of cancer, the term "prevention" refers to drug administration before the signs or symptoms of cancer occur, particularly in a subject at risk of cancer.

[0079] The term "therapeutic agent" as described herein encompasses any substance effective in the prevention or treatment of tumors (e.g., cancer) and infections (e.g., chronic infections), including chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective active agents, small molecule drugs, or immunomodulatory agents.

[0080] "Chemotherapeutic agent" includes chemical compounds effective in the treatment of cancer, including antitumor agents containing alkylating agents, antimetabolites, natural products, antibiotics, enzymes, heterologous reagents, hormones and antagonists, antiestrogen agents, antiandrogen agents, and non-steroidal antiandrogen agents, etc., but is not limited thereto. For examples of chemotherapeutic agents, see those disclosed in WO2015 / 153513 or WO2016 / 028672 or WO2015 / 138920.

[0081] As used herein, the term "immunomodulatory agent" refers to a natural or synthetic active agent or drug that suppresses or modulates the immune response. The immune response may be a humoral response or a cellular response. Immunomodulatory agents include immune checkpoint molecule inhibitors and costimulatory molecule activators.

[0082] As used herein, the term "cytotoxic agent" refers to a substance that suppresses or inhibits cell function and / or causes cell death or cell destruction. For examples of cytotoxic agents, see those disclosed in WO2015 / 153513, WO2016 / 028672 or WO2015 / 138920.

[0083] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked thereto. The term includes vectors as self-replicating nucleic acid structures and vectors that bind to the genome of the host cell into which it has been introduced. Some vectors can direct the expression of nucleic acids effectively linked thereto. Such vectors are referred to herein as "expression vectors".

[0084] The term "host cell" refers to a cell into which an exogenous polynucleotide has already been introduced, and the progeny of such a cell. A host cell includes "transformants" and "transformed cells" including the initially transformed cell and progeny derived therefrom, without regard to the number of generations. Progeny may not be identical to the parental cell with respect to the nucleic acid material and may include mutations. As used herein, mutant progeny having the same function or biological activity as those initially screened or selected from the transformed cell are included. A host cell can be any type of cell line that can be used to produce the antibody molecules of the present invention, including eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as Escherichia coli cells. A host cell includes cultured cells and also includes cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0085] "Subject / patient sample" is a collection of cells, tissues, or body fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, e.g., from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or aspirate samples, blood or any blood component, cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid, cells from a subject during pregnancy or at any stage of development. Tissue samples may contain compounds that do not occur naturally in tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. As used herein, examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial washings, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or showing tumor-like properties, and preserved tumor samples such as tumor samples fixed in formalin and embedded in paraffin or frozen tumor samples.

[0086] II. Anti-CD40 Antibodies of the Invention

[0087] The present invention provides a next-generation anti-human CD40 antibody capable of reducing or avoiding adverse events caused by conventional CD40 agonists.

[0088] The anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region. The light chain variable region comprises three complementarity-determining regions, namely LCDR1, LCDR2 and LCDR3 respectively. The heavy chain variable region comprises three complementarity-determining regions, namely HCDR1, HCDR2 and HCDR3 respectively. Here, (a) LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant with two or less or one or less amino acid changes of SEQ ID NO: 1. (b) LCDR2 comprises any one of the amino acid sequences selected from SEQ ID NO: 2, 5, 6, 7, or a variant with two or less or one or less amino acid changes of any one of the amino acid sequences of SEQ ID NO: 2, 5, 6, 7. (c) LCDR3 comprises any one of the amino acid sequences selected from SEQ ID NO: 3, 4, or a variant with two or less or one or less amino acid changes of any one of the amino acid sequences of SEQ ID NO: 3, 4. (d) HCDR1 comprises any one of the amino acid sequences selected from SEQ ID NO: 8, 12, 14, 20, or a variant with two or less or one or less amino acid changes of any one of the amino acid sequences of SEQ ID NO: 8, 12, 14, 20. (e) HCDR2 comprises any one of the amino acid sequences selected from SEQ ID NO: 9, 21, or a variant with two or less or one or less amino acid changes of any one of the amino acid sequences of SEQ ID NO: 9, 21. (f) HCDR3 comprises any one of the amino acid sequences selected from SEQ ID NO: 10, 11, 13, 15, 16, 17, 18, 19, or a variant with two or less or one or less amino acid changes of any one of the amino acid sequences of SEQ ID NO: 10, 11, 13, 15, 16, 17, 18, 19. Here, the amino acid change is an addition, deletion, or substitution of an amino acid. For example, the amino acid change is a conservative amino acid substitution.

[0089] In some embodiments, the anti-CD40 antibody of the present invention contains LCDR1 such as the amino acid sequence RSSQGIRSSLA (SEQ ID NO: 1), the amino acid sequence GX 1 SSLX 2 X 3 (SEQ ID NO: 56) contains LCDR2 such as, and the amino acid sequence QQLX 4 SFPST (SEQ ID NO: 57) contains LCDR3 such as, where X 1 is A or G, X 2 is E or L, X 3 is G or V, X 4 is N or A, and the amino acid sequence GFTX 5 GSYEMX 6 (SEQ ID NO: 58) contains HCDR1 such as, the amino acid sequence YISSX 7 GETTD (SEQ ID NO: 59) contains HCDR2 such as, and the amino acid sequence DVFFFX 8 X 9 SX 10 X 11 X 12 X 13 AYGMDV (SEQ ID NO: 60) contains HCDR3 such as, where X 5 is F, A, or P, X 6 is N or D, X 7 is S or A, X 8 is D or S, X 9 is S or P, X 10 is G or R, X 11 is D, P, S, or F, X 12 is P, N, or R, X 13 is G or H.

[0090] In some embodiments, the anti-CD40 antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein (i) the light chain variable region contains an amino acid sequence of any one of SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains an amino acid sequence of any one of SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55, or (ii) the light chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence of any one of SEQ ID NO: 23, 29, 33, 39, 53, and the heavy chain variable region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence of any one of SEQ ID NO: 25, 31, 35, 37, 41, 43, 45, 47, 49, 51, 55.

[0091] In some embodiments, the anti-CD40 antibody of the present invention comprises an immunoglobulin Fc region. In some embodiments, the C-terminus of the Fab fragment of the anti-CD40 antibody of the present invention is linked to the immunoglobulin Fc region, optionally via an amino acid linker, for example, via an amino acid linker having a length of 1 to 20 amino acids. In some embodiments, at least 90% of the amino acid linkers are glycine and / or serine amino acids. In some embodiments, the Fc region is from IgG, for example, from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region is from IgG1. In some embodiments, the Fc region is from human IgG1.

[0092] In some embodiments of the present invention, the amino acid changes described herein include amino acid substitutions, additions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, more preferably conservative substitutions.

[0093] In some embodiments, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., FRs). In some embodiments, the substitutions are conservative substitutions. A conservative substitution refers to one amino acid being replaced with another amino acid within the same class, e.g., one acidic amino acid is replaced with another acidic amino acid, one basic amino acid is replaced with another basic amino acid, or one neutral amino acid is replaced with another neutral amino acid.

[0094] In some embodiments, the anti-CD40 antibodies according to the present specification increase or decrease their degree of glycosylation by modification. Addition or deletion to the glycosylation sites of the anti-CD40 antibody can be readily achieved by modifying the amino acid sequence to produce or remove one or more glycosylation sites. When the anti-CD40 antibody contains an Fc region, the glycosylation of the Fc region can be modified. In some applications, removal of unwanted glycosylation site modifications can be useful. For example, removal of fucose modification can improve the antibody-dependent cell cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modification can be performed to regulate complement-dependent cytotoxicity (CDC).

[0095] In some embodiments, the anti-CD40 antibody of the present invention has the glycosylation site in the CH2 domain of its immunoglobulin Fc region removed. For example, the N297 residue in the CH2 domain of the human IgG Fc region is mutated to remove the glycosylation site. For example, the N297 residue is changed to Gly, Ala, Gln, Asp, or Glu, preferably the N297 residue is changed to Gln.

[0096] The anti-CD40 antibody of the present invention binds to CD40 with high affinity as measured by the ForteBio kinetic binding assay, e.g., about 10 -7 M to about 10 -10M can bind to CD40, such as human CD40, cynomolgus monkey CD40, and mouse CD40. In some embodiments, the anti-CD40 antibody of the present invention has an affinity of about 0.5×10 -8 M to 2×10 -8 M and binds to the human CD40 antigen. The anti-CD40 antibody of the present invention has cross-reactivity with cynomolgus monkey CD40 and mouse CD40.

[0097] The anti-CD40 antibody of the present invention enhances the immune response against an antigen (e.g., tumor-associated antigen (TAA)), for example, by enhancing the B cell-mediated immune response, B cell activation, and / or cytokine production, thereby enhancing the immune response against the antigen (e.g., tumor-associated antigen (TAA)).

[0098] In one embodiment, the anti-CD40 antibody of the present invention can enhance the immune response against an antigen and induce antibody-dependent cell-mediated cytotoxicity (ADCC) against CD40-expressing cells (e.g., tumor cells expressing CD40). Also, when the measured expression by CD95 increases, the anti-CD40 antibody of the present invention induces apoptosis.

[0099] In another embodiment, the anti-CD40 antibody of the present invention can enhance the immune response against an antigen without inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) of CD40-expressing cells. Therefore, the anti-CD40 antibody of the present invention can play a role in enhancing the immune response against an antigen, for example, by enhancing the B cell-mediated immune response, even against tumor cells that do not express CD40 or infectious diseases.

[0100] In one embodiment, the anti-CD40 antibody of the present invention is modified to include a constant region without effector function. For example, when the anti-CD40 antibody of the present invention includes an Fc region, the effector function of the anti-CD40 antibody of the present invention can be reduced or eliminated by mutating the glycosylation site N297 residue in the Fc region to Gly, Ala, Gln, Asp, or Glu, etc. Further, when the anti-CD40 antibody of the present invention includes an Fc region, in order to reduce or eliminate the effector function by the Fc region, the Fc region may further include a modification that reduces the binding affinity of the anti-CD40 antibody of the present invention for FcγRIIIA (CD16A). In one embodiment, the modification is in the CH2 domain of the Fc region, for example, at position 329 (EU number) of the heavy chain (e.g., P329G). In one embodiment, the anti-CD40 antibody of the present invention includes amino acid substitutions at positions 234 and 235 of the heavy chain (EU number). In one specific embodiment, the amino acid substitution is L234A and L235A (also referred to as the "LALA mutation").

[0101] In one embodiment, the anti-CD40 antibody of the present invention can enhance the immune response without relying on the binding between the antibody and the Fc receptor. For example, the anti-CD40 antibody of the present invention can exhibit effective CD40 agonist characteristics without cross-linking an Fc receptor, such as FcγR. These agonist characteristics include, for example, being determined by measuring an increase in B cell activity and / or an increase in B cell activation.

[0102] In another embodiment, the anti-CD40 antibody of the present invention enhances the binding between CD40 and CD40L (CD154) in CD40-expressing cells. In a specific embodiment, the anti-CD40 antibody of the present invention enhances the binding between soluble CD40L and CD40-expressing cells by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%. In a specific embodiment, for example, as measured by FACS, biolayer interferometry (BLI), or Biacore, the anti-CD40 antibody enhances the binding between soluble CD40L and CD40-expressing cells by at least about 50%.

[0103] In some embodiments, the anti-CD40 antibody of the present invention induces the expression of CD95 when incubated with Ramos cells. In some embodiments, the anti-CD40 antibody of the present invention increases the proliferation of B cells when incubated with human B cells. In some embodiments, the anti-CD40 antibody of the present invention increases the secretion of IL-12 and the expression of CD83 when incubated with dendritic cells.

[0104] It is expected that the anti-CD40 antibody of the present invention does not cause or causes weak adverse reactions such as dose-limiting toxicity, cytokine release syndrome (CRS), and hepatotoxicity associated with increased concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GLDH) after administration to a subject.

[0105] III. Immunoconjugate

[0106] The present invention further relates to an anti-CD40 antibody of the present invention conjugated with another substance ("immunoconjugate"). In some embodiments, the other substance is, for example, a therapeutic agent (e.g., a cytotoxic agent). Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art. For example, cytotoxic agents include, but are not limited to, radioisotopes, growth inhibitors, fragments and / or variants thereof, low molecular weight toxins such as enzymatic active toxins of bacterial, fungal, plant or animal origin, and various known antitumor or anticancer agents.

[0107] Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates may further be referred to, for example, WO2015 / 153513 or WO2015 / 138920.

[0108] The anti-CD40 antibody of the present invention may also be linked to a solid support, and the support can be used particularly for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0109] In some embodiments, the immunoconjugate is for preventing or treating a tumor. In some embodiments, the tumor is cancer. In some embodiments, the immunoconjugate is for preventing or treating an infection, such as a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, etc.

[0110] IV. Nucleic Acids of the Present Invention and Host Cells Containing the Same

[0111] In one aspect, the present invention provides a nucleic acid encoding any of the above anti-CD40 antibodies or fragments thereof or any one of its chains. In one embodiment, a vector containing the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell containing the nucleic acid or the vector is provided. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is a prokaryotic cell.

[0112] For example, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence shown in any one selected from SEQ ID NO: 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one selected from SEQ ID NO: 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55.

[0113] The present invention further covers a nucleic acid that hybridizes with the following nucleic acid under stringent conditions, or a nucleic acid encoding a polypeptide sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions as compared with the following nucleic acid: a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence shown in any one selected from SEQ ID NO: 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one selected from SEQ ID NO: 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55.

[0114] In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. The vector includes, but is not limited to, a virus, plasmid, cosmid, λ phage or yeast artificial chromosome (YAC). In one embodiment, the vector is a pcDNA3.3 vector.

[0115] When an expression vector or DNA sequence for expression is produced, the expression vector may be transfected or introduced into a suitable host cell. Various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipofection or other common techniques, can be used to achieve this purpose. The methods and conditions for culturing the produced transfected cells and recovering the produced antibody molecules are known to those skilled in the art and can be modified or optimized according to the specific expression vector and mammalian host cell used, based on the methods known in the present specification and the prior art.

[0116] In addition, by introducing one or more markers that enable the selection of transfected host cells, cells that have already stably integrated DNA into their chromosomes can be selected. The markers can provide, for example, prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may be required to optimally synthesize mRNA. These elements may include splicing signals, transcription promoters, enhancers, and termination signals.

[0117] In one embodiment, a host cell containing the polynucleotide of the present invention is provided. In some embodiments, a host cell containing the expression vector of the present invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies. Suitable host cells include prokaryotic microorganisms such as Escherichia coli. The host cell may further be a eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells may also be used as hosts. For example, a mammalian cell line engineered to be suitable for suspension growth may be used. Examples of useful mammalian host cell lines include simian kidney CV1 cells transformed by SV40 (COS-7), human fetal kidney cell lines (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), simian kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63, and Sp2 / 0. In one preferred embodiment, the host cell is a CHO cell or a 293 cell.

[0118] V. Production and Purification of the Anti-CD40 Antibody of the Present Invention

[0119] In one embodiment, the present invention provides a method for producing an anti-CD40 antibody, wherein the method comprises culturing a host cell containing a nucleic acid encoding the anti-CD40 antibody or an expression vector of the nucleic acid under conditions suitable for the expression of the nucleic acid encoding the anti-CD40 antibody, and optionally isolating the anti-CD40 antibody. In certain embodiments, the method further comprises recovering the anti-CD40 antibody from the host cell (or host cell culture medium).

[0120] To recombinantly produce the anti-CD40 antibody of the present invention, first, a nucleic acid encoding the anti-CD40 antibody of the present invention is isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced by conventional procedures, for example, using oligonucleotide probes that can specifically bind to the nucleic acid encoding the anti-CD40 antibody of the present invention.

[0121] The anti-CD40 antibody of the present invention produced as described herein can be purified by known prior art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are apparent to those skilled in the art. The purity of the anti-CD40 antibody of the present invention can be determined by any one of a plurality of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0122] VI. Method for Measuring the Activity of the Anti-CD40 Antibody of the Present Invention

[0123] The anti-CD40 antibodies according to the present specification can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a plurality of measurement methods known in the art. On the other hand, the antigen-binding activity of the anti-CD40 antibodies of the present invention is tested by known methods such as ELISA, Western blot, etc. Binding to CD40 can be measured by methods known in the art, and exemplary methods are disclosed herein. In some embodiments, surface plasmon resonance (SPR) or biolayer interferometry is used to measure the binding of the anti-CD40 antibodies of the present invention to CD40.

[0124] The present invention further provides a measurement method for identifying anti-CD40 antibodies having biological activities. The biological activities may include, for example, binding to cell surface CD40 (e.g., human CD40, monkey CD40, mouse CD40), enhancement of CD40 / CD40L binding, activation of antigen-presenting cells, induction of CD95 expression by tumor cells expressing CD40, enhancement of B cell-mediated immune responses, and the like.

[0125] The cells used in any of the above in vitro measurement methods include cell lines that naturally express CD40 or are engineered to express CD40. The cell lines engineered to express CD40 are cell lines that do not normally express CD40 and express CD40 after transfecting the cells with DNA encoding CD40.

[0126] As can be understood, any of the above measurement methods can be performed using the immunoconjugates of the present invention instead of anti-CD40 antibodies.

[0127] VII. Pharmaceutical Compositions and Pharmaceutical Preparations

[0128] In some embodiments, the present invention provides a composition comprising any anti-CD40 antibody or its immunoconjugate described herein, preferably, the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical adjuvant. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises a combination of the anti-CD40 antibody or its immunoconjugate of the present invention and one or more other therapeutic agents (e.g., a chemotherapeutic agent, a cytotoxic agent, other antibodies, an anti-infective agent, a small molecule drug or an immunomodulatory agent, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody).

[0129] In some embodiments, the composition is for preventing or treating a tumor. In some embodiments, the tumor is cancer. In some embodiments, the composition is for preventing or treating an infection, e.g., a chronic infection, e.g., a bacterial infection, a viral infection, a fungal infection, a protozoal infection, etc.

[0130] The present invention further comprises a composition (including a pharmaceutical composition or a pharmaceutical formulation) comprising an anti-CD40 antibody or its immunoconjugate, and / or a composition (including a pharmaceutical composition or a pharmaceutical formulation) comprising a polynucleotide encoding an anti-CD40 antibody. These compositions may further comprise suitable pharmaceutical adjuvants such as pharmaceutical vectors known in the art and pharmaceutical excipients including buffers.

[0131] As used herein, "pharmaceutical vehicle" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents and the like. Suitable pharmaceutical vehicles for the present invention may be sterile liquids such as oils and water, including those from petroleum, animal, vegetable or synthetic sources such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is a preferred vehicle. Also, physiological saline, aqueous dextrose and glycerin solutions may be used as liquid vehicles, particularly for injection solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, diol, water, ethanol and the like. For the use and application of excipients, reference is also made to "Handbook of Pharmaceutical Excipients", 5th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may further contain small amounts of wetting agents or emulsifying agents or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical vehicles and / or excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin and the like.

[0132] The pharmaceutical preparation containing the anti-CD40 antibody described herein can preferably be manufactured in the form of a lyophilized preparation or an aqueous solution by mixing the anti-CD40 antibody of the present invention having the desired purity with one or more optional pharmaceutical adjuvants (edited by Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (1980)).

[0133] The pharmaceutical composition or formulation of the present invention may further contain two or more active ingredients required for the specific indication to be treated, and preferably, they have active ingredients with complementary activities that do not have an adverse effect on each other. For example, it is desirable to further provide other anti-cancer active ingredients or anti-infective active ingredients, such as chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, etc. The active ingredients are appropriately combined and present in an amount effective for the intended use.

[0134] A sustained-release formulation can be manufactured. Suitable examples of the sustained-release formulation include a semi-permeable matrix containing a solid hydrophobic polymer of the anti-CD40 antibody of the present invention, and the matrix is in the form of a molded article, such as a film or a microcapsule.

[0135] VIII. Combination Product or Kit

[0136] In some embodiments, the present invention further provides a combination product comprising the anti-CD40 antibody or antigen-binding fragment of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (such as chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective active agents, small molecule drugs or immunomodulators, etc.). In some embodiments, the other antibody is, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody.

[0137] In some embodiments, the combination product is for preventing or treating a tumor. In some embodiments, the tumor is cancer or the like.

[0138] In some forms, two or more components of the combination product can be administered to a subject sequentially, separately or simultaneously.

[0139] In some embodiments, the present invention further provides a kit comprising the anti-CD40 antibody, pharmaceutical composition, immunoconjugate or combination product of the present invention, and any package insert instructing administration.

[0140] In some embodiments, the present invention further provides a pharmaceutical product comprising the anti-CD40 antibody, pharmaceutical composition, immunoconjugate, and combination product of the present invention, and optionally, the pharmaceutical product further comprises a package insert indicating administration.

[0141] IX. Use of the Anti-CD40 Antibody of the Present Invention

[0142] In one aspect, the present invention relates to a method for modulating an immune response in an individual. The method comprises modulating the immune response in a subject by administering to the subject an effective amount of the anti-CD40 antibody disclosed herein, or a pharmaceutical composition or immunoconjugate or combination product comprising said anti-CD40 antibody. In one embodiment, the therapeutically effective amount of the anti-CD40 antibody or pharmaceutical composition or immunoconjugate or combination product disclosed herein restores, enhances, stimulates or increases the immune response in the subject.

[0143] In some embodiments, the present invention relates to a method for enhancing the activity of CD40, enhancing the binding of CD40 and CD40L, and inducing the secretion of cytokines such as IL-12 in an individual, the method comprising administering to the subject an effective amount of the anti-CD40 antibody disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising the same.

[0144] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of the anti-CD40 antibody disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising the same. In some embodiments, the tumor is tumor immune escape. In some embodiments, the tumor is cancer.

[0145] In another aspect, the invention relates to a method of inducing antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of an anti-CD40 antibody disclosed herein, or a pharmaceutical composition or immunoconjugate or combination product comprising the same.

[0146] The subject can be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject is suffering from or at risk of suffering from a disease described herein (e.g., a tumor or infectious disease described herein). In some embodiments, the subject has received or is receiving other treatments, such as chemotherapy and / or radiation therapy. Alternatively or in combination, the subject is or is at risk of becoming immunocompromised due to an infection.

[0147] In some embodiments, the tumors described herein, such as cancers, include, but are not limited to, solid tumors, blood cancers, soft tissue tumors, and metastases.

[0148] Examples of solid tumors include malignant tumors, such as sarcomas and cancers (including adenocarcinomas and squamous cell carcinomas) invading the liver, lung, breast, lymph, gastrointestinal tract (e.g., colon), pancreas, urogenital tract (e.g., kidney, bladder epithelium), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers in lung cancers, small intestine cancers, and esophageal cancers. Squamous cell carcinomas include malignant tumors such as those cancers in the lung, esophagus, skin, head and neck, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, such as advanced melanoma. In one embodiment, the cancer is lymphoma, renal cell carcinoma, non-small cell lung cancer, liver cancer, pancreatic cancer, colon adenocarcinoma, breast cancer. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention.

[0149] Non-limiting examples of preferred cancers for treatment include lymphoma (e.g., diffuse large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma), breast cancer (e.g., metastatic breast cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), lung cancer (e.g., non-small cell lung cancer (NSCLC), e.g., stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (e.g., multiple myeloma), leukemia (e.g., chronic myeloid leukemia), skin cancer (e.g., melanoma (e.g., stage III or stage IV melanoma) or Merkel cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), myelodysplastic syndrome, bladder cancer (e.g., transitional cell carcinoma), kidney cancer (e.g., renal cell carcinoma, e.g., clear cell renal cell carcinoma, e.g., advanced or metastatic clear cell renal cell carcinoma), and colon cancer. Also, refractory or recurrent malignancies (e.g., pancreatic cancer) can be treated using the anti-CD40 antibodies or pharmaceutical compositions or immunoconjugates or combination products thereof described herein.

[0150] In some embodiments, the anti-CD40 antibody of the present invention binds to CD40 on the surface of B cells or antigen-presenting cells, causing a cascade effect and activating T cells to treat tumors.

[0151] In some embodiments, for tumors in which CD40 is overexpressed on the surface, the anti-CD40 antibody of the present invention treats tumors by inducing ADCC, CTL, and / or tumor cell apoptosis.

[0152] In some embodiments, the anti-CD40 antibody or immunoconjugate or composition or combination product thereof of the present invention delays the onset of the disease state and / or symptoms associated with the disease state.

[0153] In some embodiments, the prophylactic or therapeutic methods described herein further comprise co-administering to the subject or individual the anti-CD40 antibody or pharmaceutical composition or immunoconjugate or combination product disclosed herein, and one or more other therapies, e.g., treatment modalities and / or other therapeutic agents.

[0154] In some embodiments, the treatment modalities include surgery (e.g., tumor resection), radiotherapy (e.g., external particle beam therapy for three-dimensional conformal radiotherapy where the irradiation area is designed,), local irradiation (e.g., irradiation directed at a preselected target or organ) or focused irradiation). Focused irradiation can be selected from stereotactic radiosurgery, stereotactic radiosurgery by fractionated irradiation, and intensity-modulated radiotherapy. Focused irradiation can have a radiation source selected from particle beams (protons), cobalt-60 (photons), and linear accelerators (X-rays) as described in WO 2012 / 177624.

[0155] Radiotherapy can be administered by one or a combination of several methods, including but not limited to external particle beam therapy, internal radiotherapy, implant irradiation, stereotactic radiosurgery, total body radiotherapy, radiotherapy, and permanent or temporary interstitial brachytherapy.

[0156] In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective active agents, small molecule drugs, or immunomodulatory agents (e.g., activators of costimulatory molecules or inhibitors of immune checkpoint molecules).

[0157] Exemplary other antibodies include, but are not limited to, inhibitors of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-L1, anti-TIM-3, anti-CEACAM, or anti-LAG-3), antibodies that stimulate immune cells (e.g., agonist GITR antibody or CD137 antibody). Preferably, the other antibody is selected from an anti-PD-1 antibody and / or an anti-PD-L1 antibody. More preferably, the anti-PD-1 antibody is nivolumab from Bristol Myers Squibb (BMS) or pembrolizumab from Merck, and the anti-PD-L1 antibody is atezolizumab developed by Roche, avelumab jointly developed by Merck KGaA and Pfizer, or durvalumab developed by AstraZeneca.

[0158] In some embodiments, the immunomodulatory agent is an activator or agonist of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from agonists (e.g., agonist antibodies or antigen-binding fragments thereof, or soluble fusions) of the following molecules: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.

[0159] The combination therapies of the present invention cover co-administration (wherein two or more therapeutic agents are included in the same formulation or separate formulations) and separate administration. In the case of separate administration, administration of an anti-CD40 antibody or immunoconjugate of the present invention, etc., can be carried out before, simultaneously with and / or after administration of other therapies.

[0160] In one embodiment, the administration of the anti-CD40 antibody and the administration of other therapies (e.g., treatment modalities or therapeutic agents) are carried out within about one month of each other, or within about one, two or three weeks, or within about 1, 2, 3, 4, 5, or 6 days.

[0161] The anti-CD40 antibodies (and pharmaceutical compositions or immunoconjugates containing the same) of the present invention may be administered by any suitable method, including parenteral administration, intratracheal administration and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending to some extent on whether the dosing is short-term or long-term, dosing can be carried out by any suitable route, such as injection, e.g., intravenous injection or subcutaneous injection. This specification covers various dosing schedules, including single administration or multiple administrations at multiple time points, bolus infusion administration and pulse infusion, but is not limited thereto.

[0162] To prevent or treat a disease, the appropriate dosage of the anti-CD40 antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) depends on the type of disease being treated, the type of anti-CD40 antibody, the severity and progression of the disease, whether the anti-CD40 antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the anti-CD40 antibody, and the judgment of the attending physician. The anti-CD40 antibody is appropriately administered to a patient by a single treatment or a series of treatments. The dosage and treatment regimen of the anti-CD40 antibody can be determined by those skilled in the art.

[0163] As will be appreciated, any of the above prophylaxis or treatment can be carried out using the immunoconjugate or composition or combination product of the present invention instead of the anti-CD40 antibody.

[0164] X. Methods and Compositions for Diagnosis and Detection

[0165] In some embodiments, any anti-CD40 antibody according to the present specification can be used to detect the presence of CD40 in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may also relate to immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays. In some embodiments, the biological sample is blood, serum or other body fluid samples derived from an organism. In some embodiments, the biological sample contains cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

[0166] In one embodiment, a CD40 antibody used in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of CD40 in a biological sample is provided. In some embodiments, the method includes detecting the presence of CD40 protein in a biological sample. In some embodiments, CD40 is human CD40. In some embodiments, the method comprises contacting a biological sample with an anti-CD40 antibody as described herein under conditions that permit binding of the anti-CD40 antibody to CD40, and detecting whether a complex is formed between the anti-CD40 antibody and CD40. Formation of the complex indicates the presence of CD40. The method may be an in vitro or in vivo method. In one embodiment, the anti-CD40 antibody is used to select a subject suitable for treatment with the anti-CD40 antibody, e.g., where CD40 is a biomarker for selecting said subject.

[0167] In one embodiment, cancer or a tumor can be diagnosed using the anti-CD40 antibody of the present invention, e.g., to evaluate (e.g., monitor) the treatment or progression, diagnosis and / or stage of a disease (e.g., a proliferative or cancerous disease) as described herein in a subject. In some embodiments, a labeled anti-CD40 antibody is provided. The label includes, but is not limited to, a label or moiety directly detected (e.g., a fluorescent label, a chromophore label, a high electron density label, a chemiluminescent label, and a radioactive label), and a moiety indirectly detected, e.g., an enzyme or a ligand, by, for example, an enzymatic reaction or a molecular interaction. Exemplary labels are radioactive isotopes 32 P, 14 C, 125 I, 3 H and 131I. Fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidase such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide-oxidized dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, stable radicals, etc., but not limited thereto.

[0168] In some embodiments of any of the inventions according to this specification, the sample is obtained prior to treatment with an anti-CD40 antibody. In some embodiments, the sample is obtained after the cancer has already metastasized. In some embodiments, the sample is fixed with formalin and embedded in paraffin (FFPE). In some embodiments, the sample is a biopsy (e.g., core biopsy), surgical specimen (e.g., specimen from surgical resection), or fine needle aspirate.

[0169] In some embodiments, CD40 is detected prior to treatment, for example, before the start of treatment or before a certain treatment after a treatment interval.

[0170] In some embodiments, provided is a method for treating a tumor or an infection, the method comprising examining the presence of CD40 in a subject (e.g., a sample) (e.g., a subject sample containing cancer cells), thereby determining a CD40 value, comparing the CD40 value with a control value (e.g., the value of CD40 in a sample of a healthy individual), and, if the CD40 value is less than the control value, administering to the subject a therapeutically effective amount of an anti-CD40 antibody (e.g., the anti-CD40 antibodies described herein) optionally in combination with one or more other therapies, thereby treating the tumor or the infection.

[0171] As can be understood, each embodiment described in each part of the present invention, e.g., diseases, therapeutic agents, treatment modalities, and administrations, etc., is similarly suitable for embodiments of other parts of the present invention or can be combined with embodiments of other parts. Embodiments such as properties, uses, and methods suitable for anti-CD40 antibodies described in each part of the present invention are similarly suitable for compositions, conjugates, combination products, kits, etc. containing anti-CD40 antibodies.

[0172] XI. Sequences of Exemplary Anti-CD40 Antibodies of the Present Invention

[0173] The sequences of the exemplary anti-CD40 antibodies of the present invention are as shown in the following table.

[0174] [Table 1] JPEG0007692185000001.jpg88170

[0175] [Table 2] JPEG0007692185000002.jpg92170

[0176] [Table 3-1] JPEG0007692185000003.jpg222170[Table 3-2] JPEG0007692185000004.jpg226170[Table 3-3] JPEG0007692185000005.jpg225170

[0177] The following examples are described to assist in the understanding of the present invention. The examples are not intended to limit the protection scope of the present invention and should not be construed as such in any way.

[0178] Example

[0179] Example 1: Production of anti-CD40 antibody

[0180] 1.1. Production of P17-Fc antigen

[0181] The extracellular domain of human CD40 (CD40 ECD) (amino acids 21 to 193 of Uniprot: P25942) gene fragment was fused to the N-terminus of the DNA sequence of the human IgG1 Fc fragment, and the nucleotide sequence encoding the CD40 ECD-Fc fusion protein was synthesized and cloned into the polyclonal site of the pcDNA3.3 (Invitrogen) vector to obtain an expression vector expressing the CD40 ECD-Fc fusion protein, which is also referred to herein as the P17-Fc fusion protein expression vector.

[0182] The P17-Fc fusion protein expression vector was introduced into CHO-K cells (Thermo Fisher) for eukaryotic expression (for details of the production steps, refer to the relevant content of the book "Therapeutic Fc-Fusion Proteins" (Steven M. Chamow et al., pages 45 to 62, "Methods of Production for Fc-Fusion Proteins")) to obtain the P17-Fc fusion protein. The immunogenicity of the produced P17-Fc fusion protein was verified by measuring the binding status of the P17-Fc fusion protein with the anti-CD40 antibody dacetuzumab (refer to EP1885399B1, hereinafter sometimes abbreviated as CD40-Dac) and Bleslumab (refer to US20100234578, hereinafter sometimes abbreviated as CD40-Ble) by the standard ELISA method. As shown in Figure 1, the P17-Fc fusion protein showed affinity for dacetuzumab and Bleslumab.

[0183] 1.2. Biotinylation of P17-Fc fusion protein

[0184] According to the manufacturer's instructions, EZ-Link TM Sulfo-NHS-LC-biotinylation kit (EZ-Link TM Sulfo-NHS-LC-Biotinylation Kit, Thermo Fisher, product number: 21435) was used to label the calculated amount of EZ-Link Sulfo-NHS-LC-biotin onto the P17-Fc fusion protein. The amount of biotinylated P17-Fc fusion protein (also referred to herein as "P17-Fc-biotin", "biotinylated P17-Fc fusion protein") was measured by a biotin quantification kit (Thermo Fisher, 28005).

[0185] Furthermore, the conformation of the biotinylated P17-Fc fusion protein was verified by ELISA assays against dacetuzumab and Bleslumab using the same method as in Example 1.1.

[0186] As shown in Figure 2, the biotinylated P17-Fc fusion protein showed a binding similar to that of the two anti-CD40 antibodies compared to the binding of the non-biotinylated P17-Fc fusion protein to dacetuzumab and Bleslumab.

[0187] Similarly, IL-23-Fc-biotin (the IL-23 sequence source is Uniprot: Q9NPF7) was produced and used to remove phages that bind to Fc in subsequent antibody library screening.

[0188] P17-His protein (the sequence of P17 is the same as positions 21-193 of Uniprot: P25942) was produced in a similar way and has six histidine tags at its C-terminus.

[0189] 1.3. Antibody library screening

[0190] For the construction and screening method of the fully human antibody library, please refer to patent application CN202010236256.8. To obtain an anti-CD40 Fab antibody fragment displayed on phage that specifically binds to human CD40, a fully human antibody phage display library was screened using the biotinylated P17-Fc fusion protein of Example 1.1.

[0191] The specific screening method is as follows: The antibody library was screened using the magnetic bead method. Before each time of panning the fully human antibody phage display library using the biotinylated P17-Fc fusion protein of Example 1.1, the antibody library was panned with the IL-23-Fc-biotin of Example 1.1 to remove phages that bind to Fc, and phage monoclonal that can specifically bind to human CD40 protein was screened with the biotin-labeled P17-Fc fusion protein. After removing non-specifically adsorbed phages by washing, the obtained phages were collected, amplified with Escherichia coli SS320 cells, and then used for the next round of panning.

[0192] In each round of panning, the ratio of input phage to output phage was calculated and used as an indicator for CD40-specific phage enrichment.

[0193] The enrichment of CD40 by each library was also confirmed by phage library ELISA. Here, after three rounds of panning, the phages that bind to CD40 were highly enriched as shown in Figure 3.

[0194] The binding of the screened phage Fab to P17-Fc-biotin and IL23-Fc-biotin was tested. Phage clones that showed specific affinity only to P17-Fc-biotin were selected, DNA was extracted, and sequencing was performed.

[0195] One phage clone C8 containing a unique Fab sequence was obtained, and its coding nucleotide and amino acid sequences are as shown below.

[0196] [SEQ ID NO:1] JPEG0007692185000006.jpg96170

[0197] The nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of the phage C8 clone were each ligated into the pcDNA3.3 vector and expressed as Fab fragments in CHO-K cells (Thermo Fisher) and purified. The affinity of the Fab candidate molecules for P17-Fc was detected by ELISA, and the results are as shown in Figure 4.

[0198] As can be seen from Figure 4, the EC50 value for the C8 clone Fab to bind to CD40 is approximately 0.4764 μg / mL, and the EC50 value for the positive control Bleslumab mab to bind to CD40 is approximately 0.04701 μg / mL.

[0199] Furthermore, to test the affinity of the anti-CD40 Fab for CD40 expressed on the cell surface, CHO-K cells expressing human CD40 (Uniprot number P25942) on the cell surface and CHO-K cells expressing cynomolgus monkey CD40 (Uniprot number G7PG38) on the cell surface were produced. Specifically, the coding sequence of human CD40 (Uniprot number P25942) was cloned into the polylinker site of the pcDNA3.3 (Invitrogen) vector to obtain an expression vector expressing human CD40, and the expression vector expressing human CD40 was introduced into CHO-K cells (Thermo Fisher) for eukaryotic expression to obtain CHO-K cells expressing human CD40 on the cell surface (hereinafter also referred to as "CHO-K-huCD40 cells"). Similarly, CHO-K cells expressing cynomolgus monkey CD40 on the cell surface (hereinafter also referred to as "CHO-K-cynoCD40 cells") were obtained.

[0200] Inoculate CHO-K-huCD40 cells or CHO-K-cynoCD40 cells into a 96-well plate at 1.0×10 5 cells / well, and add the diluted C8 anti-CD40 Fab candidate molecule. After incubating at 4°C for 30 minutes, wash the cells and add 100 μL of APC-labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch Inc, product number: 109-136-097, allophycocyanin-labeled, F(ab') 2 fragment-specific goat anti-human IgG secondary antibody F(ab') 2 fragment (Allophycocyanin (APC) AffiniPure F(ab') 2 Fragment Goat Anti-Human IgG, F(ab') 2 fragment specific)), and incubate at 4°C for 30 minutes. Then, wash the cells and detect the binding of the C8 anti-CD40 Fab candidate molecule to the CD40 molecule expressed on the cell surface by flow cytometry (Beckman Coulter).

[0201] Figure 5A shows that different concentrations of the C8 clone anti-CD40 Fab molecule exhibit binding affinity for CHO-K-huCD40 cells, and within the C8 Fab concentration range of 3.2 μg / mL to 80 μg / mL, the binding affinity appears as C8 Fab concentration-dependent.

[0202] Figure 5B shows that the C8 clone anti-CD40 Fab molecule also exhibits binding affinity for CHO-K-cynoCD40 cells, indicating that the C8 Fab concentration used is 80 μg / mL. This reveals that the C8 clone Fab molecule has cross-reactivity with cynomolgus monkey CD40.

[0203] 1.4. Expression, purification and concentration measurement of full-length antibody

[0204] A full-length antibody construct was prepared for the C8 clone Fab candidate molecule of Example 1.3, where the sequence of human IgG1 Fc was ligated to the C-terminus of the Fab molecule heavy chain sequence.

[0205] Specifically, the obtained nucleotide sequences encoding the antibody heavy chain variable region and the nucleotide sequence encoding the antibody light chain variable region were each constructed into the eukaryotic expression vector plasmid pcDNA3.3 (Invitrogen) containing the engineered light and heavy chain constant region fragments. According to the manufacturer's instructions, the full-length heavy and light chains of the antibody were co-expressed in CHO-K cells using the ExpiCHO transient expression system (Thermo Fisher, A29133), and purified by protein A affinity chromatography to obtain the C8 clone anti-CD40 full-length antibody.

[0206] The concentration of the purified full-length antibody was measured by the A280 absorbance value, and the quality of the full-length antibody was verified by SDS-PAGE, differential scanning fluorimetry (DSF), and size exclusion chromatography (SEC). Once the quality inspection passed, it was aliquoted and stored at -80 °C. For the specific method, refer to the description in Patent Application No. CN 202010236256.8.

[0207] [SEQ ID NO: 2] JPEG0007692185000007.jpg60170

[0208] Example 2: Cell-based functional assay of anti-CD40 antibody

[0209] 2.1. CD40 binding characteristics and species cross-reactivity of candidate anti-CD40 full-length antibodies

[0210] To confirm the binding activity and cross-reactivity of the full-length anti-CD40 antibody produced in Example 1.4 of the present invention to CD40, CHO-K-huCD40 cells or CHO-K-cynoCD40 cells were seeded at 1.0×10 5Cells were seeded at 1.0×10 cells / well in a 96-well plate. 100 μL of the diluted anti-CD40 candidate antibody produced in Example 1.4 was added to the 96-well plate. After incubation at 4°C for 30 minutes, the cells were washed, and 100 μL of an APC-labeled anti-human IgG secondary antibody (Allophycocyanin (APC) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch Inc, catalog number: 109-135-098) was added, and further incubated at 4°C for 30 minutes. Then, the cells were washed, and the binding of the full-length anti-CD40 antibody to the CD40 molecules expressed on the cells was detected by flow cytometry. The results are as shown in FIGS. 6A and 6B.

[0211] As can be seen from FIGS. 6A and 6B, after constructing a full-length antibody structure against the C8 clone Fab fragment, the C8 clone anti-CD40 full-length antibody still retained the affinity of the C8 clone Fab fragment for human CD40 and the cross-reactivity for cynomolgus CD40.

[0212] Similar to Example 1.3, CHO-K cells expressing full-length mouse CD40 (Uniprot number P27512) (hereinafter also referred to as "CHO-K-mouseCD40 cells") were produced. Using the CHO-K-mouseCD40 cells, the cross-reactivity of the candidate C8 full-length anti-CD40 antibody produced in Example 1.4 with mouse CD40 was tested. CHO-K-mouseCD40 cells were seeded at 1.0×10 5 cells / well in a 96-well plate. 100 μL of the diluted anti-CD40 candidate antibody and two control antibodies (dacetuzumab, APX005) were added to the 96-well plate respectively. After incubation at 4°C for 30 minutes, the cells were washed, and an FITC-labeled anti-human IgG Fcγ secondary antibody (affinity-purified F(ab') 2 fragmented goat anti-human IgG secondary antibody (AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, specific for Fcγ fragment (Jackson Immunoresearch, catalog number: 109-006-098) was added and incubated at 4°C for 30 minutes. Then the cells were washed and tested by flow cytometry. The results are as shown in Figure 6C.

[0213] As can be seen from Figure 6C, the full-length antibody molecule of clone C8 shows significant binding activity to mouse CD40, while neither the control antibody dacetuzumab nor APX005 has cross-reactive activity against mouse CD40.

[0214] 2.2. Effect of candidate anti-CD40 antibodies on the binding of CD40 and CD40L

[0215] To test the effect of the full-length anti-CD40 antibody produced in Example 1.4 on the binding of CD40 and CD40L, CHO-K cells expressing human CD40 (i.e., CHO-K-huCD40 cells) were seeded in a 96-well plate at 1.0×10 5 cells / well. 100 μL of diluted anti-CD40 full-length antibody or isotype control (i.e., purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502)) was added to the 96-well plate. After incubation at 4°C for 30 minutes, the cells were washed.

[0216] According to the method described in Example 1, CD40L (ACRO Biosystems, product number CDL-H5248) was biotinylated, the biotinylated CD40L protein was diluted to 200 ng / mL, and 100 μL of the diluted, biotinylated CD40L (also referred to herein as CD40L-biotin) was added to a 96-well plate. After incubating at 4°C for 30 minutes, the cells were washed, and then 100 μL of PE-labeled streptavidin (eBioscience product number 12-4317-87) was added and incubated at 4°C for an additional 30 minutes. Then, the cells were washed and tested by flow cytometry.

[0217] As shown in Figure 7, candidate antibody clone C8 exhibits the activity of enhancing the binding between CD40 and CD40L when the concentration is 0.01 - 20 μg / mL, and is an anti-CD40 antibody that has the potential to become a CD40 agonist type. The control antibody Bleslumab mab significantly inhibits the binding between CD40 and CD40L, and dacetuzumab does not affect the binding between CD40 and CD40L at low concentrations and inhibits the binding between CD40 and CD40L at high concentrations.

[0218] 2.3. Assay for activation of candidate anti-CD40 antibody-mediated immature dendritic cells

[0219] Immature dendritic cells (iDC) are transformed into mature DC cells after being activated by anti-CD40 antibodies. In this example, the activation activity of candidate antibody-mediated iDC was evaluated by testing the secretion of IL-12 cytokine by mature DC cells, the expression of CD83 marker, etc.

[0220] Monocytes produced from human peripheral blood were developed into iDC cells in RPMI 1640 medium supplemented with 1000 U / mL rhGM-CSF (PeproTech product number: 300-03-50) and 500 U / mL rhIL-4 (PeproTech product number: 200-04-50) and supplemented with 10% FCS, and 5.0×10 4Individual iDC cells were seeded in 6-well plates at a density of [number] cells / well. Dilutions of the candidate anti-CD40 full-length antibody, each control antibody, and CD40L (ACRO Biosystems, product number CDL-H5248) were added to the iDC cells, and the cells were cultured for three days. Then, the culture supernatants and cells were collected.

[0221] For the collected culture supernatants, IL-12 secretion was measured by ELISA or flow cytometry, and for the collected cells, the expression of the CD83 marker was measured.

[0222] As shown in FIGS. 8A and 8B, the C8 clone full-length antibody exhibited significant and dose-dependent iDC activation activity. In this regard, similar to dacetuzumab, the C8 clone full-length antibody is a weak agonist of CD40. CD40L as a control resulted in a large amount of IL-12 secretion.

[0223] 2.4. CD95 Induction in Candidate Anti-CD40 Antibody-Mediated Ramos Cells

[0224] Ramos cells are human lymphoma cells that naturally express the CD40 molecule on the cell surface. To test whether the anti-CD40 antibody binds to CD40 on Ramos cells and activates the downstream signaling pathway of CD40, Ramos cells (ATCC No.: CRL-1596) were seeded in wells of microtiter plates containing RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (hereinafter also referred to as "complete RPMI 1640 medium") at a density of 1.0×10 5 cells / well. Diluted anti-CD40 antibody or CD40L (10 μg / mL in the first well, serially diluted 2-fold, with four concentration spots, and the concentration details are shown in FIG. 9) were added to the Ramos cells, and the cells were incubated overnight at 37°C. Then, the CD95 expression induced on the surface of Ramos cells was measured by flow cytometry.

[0225] As shown in Fig. 9, compared with hIgG1 as a negative control, the full-length C8 clone antibody showed dose-dependent CD95 induction activity and weak agonist activity.

[0226] Example 3: In Vivo Efficacy Test of Anti-CD40 Antibody

[0227] 3.1. Tumor Growth Inhibition of Ramos Cells in a Mouse CDX Model by an Anti-CD40 Agonist Antibody

[0228] The tumor cell line xenograft (CDX, cell derived xenograft) model is a tumor model constructed by transplanting a tumor cell line into the body of a nude mouse or an NSG mouse. In this example, a mouse CDX model was constructed by transplanting Ramos cells subcutaneously into mice, and the antitumor effect of a candidate anti-CD40 antibody in the mouse CDX model was tested.

[0229] On day 0 (starting from the day of inoculating tumor cells, defined as day 0, and the next day as day 1), 5 million human lymphoma Ramos cells were subcutaneously injected into six-week-old female nude mice (Vital River Balb / c Nude). When the average tumor volume reached 250 mm 3 , at 8 animals per group, all mice were administered 0.2 mg / kg, 1 mg / kg, or 5 mg / kg (hereinafter, mg / kg is also abbreviated as mpk) of the C8 anti-CD40 full-length antibody by intraperitoneal injection. The administration was three times a week (TIW) and continued for two weeks. Before each injection, the tumor volume was measured, and the tumor weight was measured two weeks after the first administration. The final tumor volume and tumor weight are shown in Fig. 10A and Fig. 10B, respectively, and the change in tumor volume is shown in Fig. 10C.

[0230] Compared with the control antibody APX005M (referred to as APX or APX005 herein) (see US20170246297, Apexigen), the C8 anti-CD40 full-length antibody showed a similar antitumor effect.

[0231] The TGI (tumor growth inhibition rate) of the antibodies in each group is as shown in Table 4.

[0232] [Table 4] JPEG0007692185000008.jpg54170

[0233] 3.2. Tumor growth inhibition by anti-CD40 agonist antibody in a mouse model of MC-38 cells

[0234] MC38 is a mouse colon adenocarcinoma cell line that highly expresses mouse p53 protein (mp53) and does not express the CD40 molecule.

[0235] In a mouse subcutaneous transplantation model of MC-38 cells, the antitumor effect of the candidate anti-CD40 antibody was tested.

[0236] One million mouse colon adenocarcinoma cells (MC-38 cells) (China National Experimental Cell Resource Sharing Platform, catalog number 3111C0001CCC000523) were subcutaneously injected into the axilla of six-week-old female mice (Vital River C57BL / 6). When the average tumor volume reached 80-100 mm 3 the mice (8 per group) were administered 1.5 mg / kg, 5 mg / kg or 15 mg / kg (hereinafter, "mg / kg" is also abbreviated as "mpk") of the C8 anti-CD40 antibody by intraperitoneal injection. The administration was three times a week (TIW) and continued for three weeks. Before each injection, the tumor volume was measured, and the tumor weight was measured three weeks after the first administration. The final tumor weight is shown in Figure 10D.

[0237] As can be seen from Figure 10D, compared with the negative control PBS, the anti-CD40 agonist antibody C8 molecule showed an antitumor effect with a concentration gradient.

[0238] Example 4. Affinity variants of the C8 clone anti-CD40 antibody

[0239] To obtain modifications to the CD40 antigen affinity by the full-length antibody of the C8 clone, this example performed further affinity variant design and functional assays on the full-length antibody of the C8 clone.

[0240] 4.1 Construction of an affinity variant phage display library

[0241] First, for C8-WT-VL and C8-WT-VH obtained in Example 1.3, the CDRs and framework regions were defined using the AbM numbering system, and a series of primers were designed to introduce single-point or consecutive triple-point mutations to each CDR, thereby constructing an affinity variant phage display library.

[0242] 4.2 Screening of the affinity variant phage display library

[0243] The screening of the affinity variant phage display library was performed using immunotube screening (i.e., solid-phase screening). Immunotubes were coated with the antigen protein P17-His (produced using a similar method as in Example 1), and the immunotubes that bind to the antigen and the affinity variant phage display library were incubated, washed, and eluted through the panning process. After 2 - 3 rounds of panning, specific monoclonal antibodies against the antigen can be highly enriched. For the specific implementation method of screening C8 affinity variants from the phage display library, refer to the immunotube screening method in Patent Application No. 202010236256.8.

[0244] After screening C8 affinity variants from the affinity variant phage display library, at the ELISA level, many candidate antibodies that can bind to the P17-His antigen with high specificity were obtained. According to the binding affinity of the Fab molecules, the first 11 candidate molecules with the highest binding affinity were selected from them to construct full-length antibodies. Here, the sequence of human IgG1 Fc silent (N297Q mutation) (i.e., huIgG1(N297Q)) was ligated to the heavy chain sequence of the Fab molecule to obtain a fully human IgG1(N297Q) antibody with both light chains being of the κ type.

[0245] 4.3. Production of full-length antibodies of affinity variant candidate clones

[0246] For the specific method, please refer to "Expression, purification and concentration measurement of full-length antibodies" in Example 1.4. C8-1 antibody, C8-2 antibody, C8-3 antibody, C8-4 antibody, C8-5 antibody, C8-6 antibody, C8-7 antibody, C8-8 antibody, C8-9 antibody, C8-10 antibody, and C8-11 antibody were obtained. The nucleotide and amino acid sequences of the variable regions of the C8 parental antibody and each variant antibody are as shown in Table 3.

[0247] 4.4. Assay for non-specific binding of affinity variant candidate antibodies

[0248] First, the relationship between the non-specific binding of the obtained affinity variant candidate antibodies to different cells and the antibody concentration used was detected. Since HEK293 cells and CHO-K cells are both cells that do not naturally express the CD40 molecule on the cell surface, antibodies that specifically bind to CD40 do not specifically bind to HEK293 cells and CHO-K cells. The positive control used in this experiment is the F4AM4 antibody described in Patent CN202010825379.5. The F4AM4 antibody is a fully human antibody, and its antibody light and heavy chain sequences are as shown in F4AM4-LC and F4AM4-HC in Table 5, respectively.

[0249] In the experiment, HEK293 (ATCC: CRL-3216) and CHO-K (ATCC: CCL-61) cells were 1.0×10 5Cells were seeded in 96-well plates at a density of [quantity] cells / well. 100 μL of serially diluted anti-CD40 candidate antibody produced in Example 4.3 was added to the 96-well plates. After incubation at 4°C for 60 minutes, the cells were washed five times with FACS buffer (PBS + 5% FBS + 2% BSA), 100 μL of PE-labeled anti-human IgG-Fc secondary antibody (Goat F(ab')2 Anti-Human IgG-Fc(PE), pre-adsorbed, Abcam, product number: ab98596) was added, and incubation was continued at 4°C for an additional 30 minutes. The cells were then washed and tested by flow cytometry.

[0250] As shown in FIGS. 11A and 11B, the high-concentration C8-8 clone showed non-specific binding on both HEK293 and CHO-K cells. C8-6 showed some non-specific binding to HEK293 at a high concentration (100 μg / mL), but none of the other candidate clones and IgG1 showed non-specific binding to these two cell lines. Therefore, in subsequent experiments, the following tests were performed using candidate molecules that did not bind non-specifically.

[0251] [Table 5] JPEG0007692185000009.jpg97170

[0252] 4.5. Cell-based functional assay of affinity variant candidate antibodies

[0253] 4.5.1. CD40 binding and cross-reactivity of affinity variant candidate antibodies

[0254] To confirm the binding activity and cross-reactivity of the affinity variant antibodies of Example 4.3, CHO-K cells (CHO-K-huCD40 cells) artificially constructed to express full-length human CD40 were seeded at a density of 1.0×10 5Cells were seeded at 1.0×10

[0255] As shown in FIGS. 12A and 12B, after constructing full-length antibody constructs against the affinity variant Fab, all candidate antibodies showed affinity for human CD40 and were equivalent to the control antibody APX005 (since APX005 showed good therapeutic effects against pancreatic cancer in the results of clinical stage II, in the experiments related to the C8 antibody affinity variant, the control antibody was set to APX005, and the APX005 sequence source is Apexigen Inc. US20120301488 and is manufactured by Sanyou Biologics).

[0256] CHO-K cells (CHO-K-mouseCD40 cells) expressing artificially constructed full-length mouse CD40 (Uniprot#P27512) were used to test the cross-reactivity between the affinity variant candidate antibody produced in Example 4.3 and mouse CD40. CHO-K cells expressing mouse CD40 were seeded at 1.0×10 5 cells / well in a 96-well plate. 100 μL of the diluted affinity variant candidate antibody and the control antibody (APX005) were added to the 96-well plate respectively. After incubating at 4° C. for 30 minutes, the cells were washed, and FITC-labeled anti-human IgG Fcγ (affinity-purified F(ab') 2 fragmented goat anti-human IgG secondary antibody (AffiniPure F(ab') 2Fragment Goat Anti-Human IgG, Fcγ fragment specific) from Jackson Immunoresearch (product number: 109-006-098) was added and incubated at 4°C for 30 minutes. Then the cells were washed and tested by flow cytometry.

[0257] As shown in FIGS. 12C and 12D, C8-6 was shown to have better mouse CD40 antigen-binding activity than the parental C8-WT. C8-1, C8-2, C8-7, C8-8, C8-9, C8-10, and C8-11 all showed binding activity to the mouse CD40 antigen.

[0258] 4.5.2. CD95 induction in Ramos cells mediated by affinity variant candidate antibodies (in the presence of a crosslinking agent, i.e., the Crosslinking effect exists)

[0259] To test whether the affinity variant candidate antibody binds to CD40 on Ramos cells and activates the downstream signaling pathway of CD40 in the presence of a crosslinking agent, Ramos cells (ATCC No.: CRL-1596) were seeded at 1.0×10 5 cells / well in complete RPMI 1640 medium. Diluted affinity variant candidate antibodies, control antibody APX005, and CD40L (20 μg / mL in the first well, serially diluted 3-fold, with 8 concentration spots) were added to the Ramos cells, along with a crosslinking agent (affinity-purified F(ab') 2 fragmented goat anti-human IgG secondary antibody that can specifically react with the Fcγ fragment (AffiniPure F(ab') 2 Fragment Goat Anti-Human IgG, Fcγ fragment specific) from Jackson Immunoresearch (product number: 109-006-098) was added, with the crosslinking agent concentration at 20 μg / mL, and incubated overnight at 37°C. Then CD95 expression was measured by flow cytometry.

[0260] As shown in FIGS. 13A and 13B, all of the affinity variant antibodies showed CD95-inducing activity equivalent to that of APX005, and there was no significant difference.

[0261] 4.5.3. CD95 Induction in Ramos Cells Mediated by Affinity Variant Candidate Antibodies (Without Crosslinking Agent, i.e., No Crosslinking Effect)

[0262] To test whether the affinity variant candidate antibody binds to CD40 on Ramos cells and activates the downstream signaling pathway of CD40 in the absence of a crosslinking agent, Ramos cells (ATCC No.: CRL-1596) were seeded at 1.0×10 5 cells / well in complete RPMI 1640 medium. Diluted affinity variant candidate antibodies, control antibody APX005, and CD40L (20 μg / mL in the first well, serially diluted 3-fold, with 8 concentration spots) were added to the Ramos cells and incubated overnight at 37°C. Then, CD95 expression was measured by flow cytometry.

[0263] As shown in FIGS. 13C and 13D, all affinity variant antibodies showed weaker CD95 induction activity than APX005 and CD40L. Here, C8-2, C8-6, C8-9, C8-10, and C8-11 showed CD95 induction activity approximately 10-fold weaker than APX005. Thus, in the absence of a crosslinking agent, the candidate molecules of the present invention showed significantly lower CD40 molecule activation ability compared to APX005, weakly agonized the CD40 molecule, and were shown to be expected to have significantly lower toxic side effects than APX005. The main adverse event of the anti-CD40 agonist-type antibody currently undergoing clinical trials is hepatotoxicity. A large amount of FcR receptors are expressed on the surface of liver cells. On the other hand, the agonist-type antibody (e.g., APX005) used in clinical trials usually has normal Fc function and can form a crosslinking effect with the FcR receptor on the surface of hepatocytes, non-specifically activating immune cells expressing CD40 on the surface of hepatocytes, thus causing hepatotoxicity and having a low therapeutic index. By analyzing the mechanism of the crosslinking effect, it was found that the crosslinking effect causing hepatotoxicity occurs due to the binding of the Fc receptor. If the crosslinking effect by the Fc receptor can be controlled and the activation effect at the lesion site can be significantly enhanced, the hepatotoxicity can be controlled and the therapeutic index can be improved. A higher requirement has been put forward for the difference in agonist activity of antibody drug molecules with or without the crosslinking effect.

[0264] In order to maximize the ratio of killing efficacy to toxicity, the present invention expands the degree of the maximum difference in agonist effects in the presence and absence of the Crosslinking effect, that is, when the crosslinking effect is formed, a significant immune activation reaction occurs, and when there is no crosslinking effect, it is desired to improve the therapeutic index by not generating or generating only slightly the immune activation effect. As can be seen from this example, the APX005 molecule has a small difference in immune cell agonist activity with or without a crosslinking agent, that is, the ratio of efficacy to toxicity after the final administration is small. On the other hand, the candidate antibody of the present invention can maximize the ratio of efficacy to toxicity with or without a crosslinking agent, and can maximize the activation of the killing effect of immune cells in the tumor microenvironment, avoiding toxic side effects such as hepatotoxicity caused by non-specific activation of immune cells at non-tumor locations. If a bispecific antibody is constructed with the candidate antibody of the present invention, by utilizing this feature, specific and high-intensity activation of immune cells at the lesion location can be achieved, and low agonist activity can be achieved at locations such as hepatocytes where the crosslinking effect cannot be formed, thereby achieving the purpose of specifically killing tumor cells without causing adverse events such as hepatotoxicity, effectively improving the therapeutic index, and improving safety.

[0265] 4.5.4. Production of a stable transfected cell line of Jurkat NF-κB luciferase reporter gene

[0266] First, using an electroporator (Invitrogen, NeonTM Transfection System, MP922947), the vector pGL4.32[Luc2p / NF-κB-RE / Hygro] plasmid (Promega, product number E8491) was transfected into Jurkat cells (ATCC(R) TIB-152 TM) was subjected to electroporation. After electroporation, the obtained cells were each transferred to RPMI 1640 medium (Hyclone, SH30243.01) containing 10% (v / v) FBS (Gibco, 15140-141) but no antibiotics, and the cells were inoculated into a cell culture dish of a 6-well plate and cultured for 48 hours. Next, the cells were dispensed into a 96-well cell culture plate at an average density of 1500 cells / well, and hygromycin B (Source BioScience, S160J7) with a final concentration of 500 μg / mL was added for screening. The growth status of cell line clones was observed for about 2 to 3 weeks, and the cell lines that formed clones were selected and transferred to a 24-well plate. After cell culture was expanded, some clones were taken and transferred to a 96-well white bottom plate (Corning, 3610), and stimulated with phorbol ester (use concentration 10 ng / ml) and ionomycin (use concentration 1 nM). At 37°C, 5% CO 2 After culturing in an incubator at 6 h, Bright-Lite substrate (Vazyme, DD1204-03) was added, and after the signal value was read by a microplate reader (Molecular Devices: Spectramax i3x), the expression levels of different clone NF-κB were evaluated to obtain a Jurkat cell line with high expression of the NF-κB gene. The stable transfected cell clones of the Jurkat NF-κB luciferase reporter gene with high expression level of NF-κB were cryopreserved.

[0267] 4.5.5. Construction of Jurkat CD40 / NF-κB luciferase reporter gene cell line

[0268] In this example, the constructed Jurkat CD40 / NF-κB luciferase reporter gene cell line is for screening candidate antibody molecules that can activate the CD40 downstream signal activity. Using the stable transfected cell line of the Jurkat NF-κB luciferase reporter gene produced in Example 4.5.4, in addition, the full-length expression gene sequence of CD40 (Uniprot Gene ID: P25942) was stably transfected, and monoclonal cell lines were screened. By adding CD40L recombinant protein to this cell line culture system, binding to CD40 activates the transcription and expression of the intracellular NF-κB luciferase reporter gene, and a luciferase catalytic substrate is added to produce a fluorescence signal. The production process of the Jurkat CD40 / NF-κB luciferase reporter gene cell line is as follows: Construction of a plasmid expressing full-length human CD40 (Met1-Gln277): A DNA fragment containing the human CD40 protein was synthesized by gene synthesis technology and cloned into the pLVX-Puro expression vector (Clontech, 632164). Escherichia coli was introduced by transformation. Escherichia coli monoclonal was selected for sequencing to obtain an accurate plasmid clone, the plasmid was extracted, and sequencing was performed again for confirmation. Electroporation: The Jurkat cells produced in Example 4.5.4 were cultured using Gibco's RPMI 1640 serum-free medium (product number: 11875085). One day before electroporation, the cells were 2×10 5Subcultured at [unit not specified] / mL, and the next day, the constructed plasmid was transformed into the stably transfected cell line of Jurkat NF-κB luciferase reporter gene using Invitrogen's electroporation kit (product number: MPK10096) and electroporator (product number: MP922947). The cells after electroporation were transferred to RPMI 1640 medium and cultured in a cell incubator at 37°C for 48 h. Seeding of electroporated cells: The electroporated Jurkat cells were seeded into a 96-well plate at 1000 cells / well, puromycin with a final concentration of 2 μg / mL was added, and the cells were cultured in a carbon dioxide incubator at 37°C. After 14 days, the RPMI 1640 medium supplemented with 2 μg / mL puromycin was replenished. Clone selection, cell expansion culture, and FACS identification: Single cell clones grown in a 96-well plate were collected, transferred to a 24-well culture plate for continuous expansion culture, and then the cell line successfully stably transfected with human CD40 was identified by FACS.

[0269] 4.5.6. Activity of candidate antibodies of affinity variants to activate the CD40 downstream NF-κB luciferase reporter gene signal (in the presence of a crosslinking agent, i.e., the Crosslinking effect exists)

[0270] In this example, to test whether the candidate antibody of the affinity variant has the function of activating the CD40 signaling pathway in the presence of a crosslinking agent, the Jurkat CD40 / NF-κB luciferase reporter gene cell line produced in Example 4.5.5 was used as a material to detect the ability of the candidate molecule to activate the expression of the downstream NF-κB luciferase reporter gene by binding to CD40. The specific implementation form is as follows: The parental C8 molecule and the candidate antibody of the affinity variant, the control antibody APX005 (10 μg / mL in the first well, serially diluted 3-fold, with 8 concentration spots) were serially diluted in RPMI 1640 medium, and a 10 μg / mL crosslinking agent (affinity-purified F(ab') that can specifically react with the Fcγ fragment) 2Fragment Goat Anti-Human IgG, Fcγ fragment specific) from Jackson Immunoresearch (Catalog No.: 109-006-098) was added for premixing. The premixed solution of the antibody and the crosslinker was added to a 96-well plate at 50 μL per well. The Jurkat CD40 / NF-κB luciferase reporter gene cell line was revived, and cells in good growth condition after 2 - 4 passages were used in the experiment. The cells were washed with RPMI 1640 medium and resuspended. After counting, the cell density was adjusted to 2×10 2 cells / mL. Then, 50 μL per well was added to the 96-well cell culture plate containing the premixed solution of the antibody and the crosslinker, and the plate was placed in a cell incubator at 37°C and incubated for 6 h. After the culture was completed, 30 μL of the luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well. After shaking for 5 min, the fluorescence value of the 96-well plate was detected. 6 As shown in FIGS. 14A, 14B, 14C, and 14D, in the presence of the crosslinker, both the parental C8 molecule and all the affinity variant antibodies showed an activity equivalent to that of APX005 in activating the CD40 downstream NF-κB luciferase reporter gene signal, and there was no significant difference.

[0271]

[0272] 4.5.7. Activity of candidate antibodies of affinity variants in activating the CD40 downstream NF-κB luciferase reporter gene signal (in the absence of a crosslinker, i.e., no Crosslinking effect)

[0273] In the examples, to test whether the candidate antibodies of affinity variants have the function of activating the CD40 signaling pathway in the absence of a crosslinker, the Jurkat CD40 / NF-κB luciferase reporter gene cell line was used as a material to detect the ability of the candidate molecules to activate the expression of the downstream NF-κB luciferase reporter gene by binding to CD40. The specific embodiments are as follows.​

[0274] The parental C8 molecule and each affinity variant candidate antibody, and the control antibody APX005 (10 μg / mL in the first well, serially diluted 3-fold, with 8 concentration spots) were serially diluted in RPMI 1640 medium. The diluted candidate antibodies and the control antibody were added to a 96-well plate at 50 μL per well. The Jurkat CD40 / NF-κB luciferase reporter gene cell line was revived, and cells that had been passaged 2 - 4 times and had a good growth state were used in the experiment. The cells were washed with RPMI 1640 medium and resuspended. After counting, the cell density was adjusted to 2×10 6 cells / mL, and added to the 96-well cell culture plate with antibodies at 50 μL per well, and placed in a cell incubator at 37°C for 6 h of incubation. After the culture was completed, 30 μL of the luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, shaken for 5 min, and then the fluorescence value of the 96-well plate was detected.

[0275] As shown in FIGS. 15A, 15B, 15C, and 15D, in the absence of a crosslinking agent, APX005 showed high activity in activating the CD40 downstream NF-κB luciferase reporter gene signal. However, among the affinity mutant candidate antibodies, except that C8-7 and C8-9 had a certain degree of activation ability, none of the other candidate antibodies had activation ability. These molecules are predicted to weakly agonize the CD40 molecule and have significantly lower toxic side effects than APX005. Comparing the activation status of the CD40 signaling pathway by each candidate molecule in FIGS. 14A - 14D and FIGS. 15A - 15D, whether there is a Crosslinking effect or not, the situation where the parental C8 molecule and each affinity variant candidate molecule activate the CD40 signaling pathway is substantially consistent with the B cell activation status in FIGS. 13A - 13D, which also verified the results of the B cell (Ramos cell) activation test from the molecular mechanism. From the test results, it was found that the B cell activation by the anti-CD40 antibody in the present invention is formed by the activation of the CD40 signaling pathway.

[0276] 4.6. Kinetic Detection of the Affinity of Candidate Antibodies of Affinity Variants

[0277] In this example, the Fortebio Octet RED96 instrument was used to detect the affinities of the C8 parental antibody (C8-WT) and the affinity variant antibodies for the human CD40 antigen (P17-His).

[0278] Preparation of materials: Weighed 1 g of BSA, measured 500 μL of Tween 20, added them to 1000 mL of 1×PBS, and mixed uniformly. After filtration, it was dispensed and stored. Aspirated 0.1 mL of 0.1 M glycine solution at pH 2.0, added 0.9 mL of ultrapure water, and mixed uniformly. Diluted the antibody to 10 μg / mL with KB buffer, and diluted the antigen with KB buffer to a series of concentration gradients of 40 nM, 20 nM, 10 nM, 5 nM, and 0 nM in sequence.

[0279] Procedure of the experiment: After shading and pre-wetting the sensor (Protein A sensor) for at least 10 min, the test for the sample plate (GreinerBio, PN655209) was started. If there was no error in the test, it was carried out according to the preset procedure. Here, 200 μL / well of KB buffer was added to columns 1, 10, and 12 of sample plate 1, 0.01 M glycine solution at pH 2.0 was added to column 11, the prepared sample solutions were added to columns 2 - 8 (one sample was added to five wells), and P17-His was added to column 9 in descending order of concentration. The details of the data results are shown in Table 6.

[0280] [Table 6] JPEG0007692185000010.jpg89170

[0281] As can be seen from Table 6, the C8 parental antibody (C8-WT) and the affinity variant antibodies bind to the human CD40 antigen with an affinity of approximately 0.5×10 -8 M - 2×10 -8 M. Here, all the affinity variant antibodies have improved affinity compared to the affinity of the C8 parental antibody (C8-WT) and are affinity mature antibodies.

Sequence Listing Free-Text

[0282] SEQ ID NOs: 1 to 55: Antibody sequences SEQ ID NO: 56: (2)..(2) Xaa may be Ala or Gly SEQ ID NO: 56: (6)..(6) Xaa may be Glu or Leu SEQ ID NO: 56: (7)..(7) Xaa may be Gly or Val SEQ ID NO: 57: (4)..(4) Xaa may be Asn or Ala SEQ ID NO: 58: (4)..(4) Xaa may be Phe, Ala or Pro SEQ ID NO: 58: (10)..(10) Xaa may be Asn or Asp SEQ ID NO: 59: (5)..(5) Xaa may be Ser or Ala SEQ ID NO: 60: (6)..(6) Xaa may be Asp or Ser SEQ ID NO: 60: (7)..(7) Xaa may be Ser or Pro SEQ ID NO: 60: (9)..(9) Xaa may be Gly or Arg SEQ ID NO: 60: (10)..(10) Xaa may be Asp, Pro, Ser or Phe SEQ ID NO: 60: (11)..(11) Xaa may be Pro, Asn or Arg SEQ ID NO: 60: (12)..(12) Xaa may be Gly or His SEQ ID NOs: 61, 62: Antibody sequences

Claims

Claim 1 An anti-CD40 antibody or an antigen-binding fragment of said anti-CD40 antibody, comprising a light chain variable region containing three complementarity-determining regions, which are LCDR1, LCDR2, and LCDR3, respectively, and a heavy chain variable region containing three complementarity-determining regions, which are HCDR1, HCDR2, and HCDR3, respectively, wherein said anti-CD40 antibody is a WT antibody (LCDR1 of SEQ ID NO: 1, LCDR2 of SEQ ID NO: 2, LCDR3 of SEQ ID NO: 3, HCDR1 of SEQ ID NO: 8, HCDR2 of SEQ ID NO: 9, and SE contains the HCDR3 of SEQ ID NO: 10), the first antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 2, the LCDR3 of SEQ ID NO: 4, the HCDR1 of SEQ ID NO: 8, the HCDR2 of SEQ ID NO: 9, and the HCDR3 of SEQ ID NO: 10), the second antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 2, the LCDR3 of SEQ ID NO: 3, the HCDR1 of SEQ ID NO: 8, the HCDR2 of SEQ ID NO: 9, and the HCDR3 of SEQ ID NO: 11), the third antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 5, the LCDR3 of SEQ ID NO: 3, the HCDR1 of SEQ ID NO: 12, the HCDR2 of SEQ ID NO: 9, and the HCDR3 of SEQ ID NO: 10), the fourth antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 2, the LCDR3 of SEQ ID NO: 3, the HCDR1 of SEQ ID NO: 8, the HCDR2 of SEQ ID NO: 9, and the HCDR3 of SEQ ID NO: 13), the fifth antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 6, the LCDR3 of SEQ ID NO: 3, the HCDR1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 9, and the HCDR3 of SEQ ID NO: 10), the sixth antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 2, the LCDR3 of SEQ ID NO: 3, the HCDR1 of SEQ ID NO: 8, the HCDR2 of SEQ ID NO: 9, and the HCDR3 of SEQ ID NO: 15), the seventh antibody (contains the LCDR1 of SEQ ID NO: 1, the LCDR2 of SEQ ID NO: 2, the LCDR3 of SEQ ID NO: 3, the HCDR1 of SEQ ID NO: 8, the HCDR2 of SEQ ID NO: 9, and SEQ ID No. 16 HCDR3), the 8th antibody (SEQ ID NO: 1 LCDR1, SEQ ID NO: 2 LCDR2, SEQ ID NO: 3 LCDR3, SEQ ID NO: 8 HCDR1, SEQ ID NO: 9 HCDR2, and SEQ ID NO: 17 HCDR3), the 9th antibody (SEQ ID NO: 1 LCDR1, SEQ ID NO: 2 LCDR2, SEQ ID NO: 3 LCDR3, SEQ ID NO: 8 HCDR1, SEQ ID NO: 9 HCDR2, and SEQ ID NO: 18 HCDR3), the 10th antibody (SEQ ID NO: 1 LCDR1, SEQ ID NO: 2 LCDR2, SEQ ID NO: 3 LCDR3, SEQ ID NO: 8 HCDR1, SEQ ID NO: 9 HCDR2, and SEQ ID NO: 19 HCDR3), and the 11th antibody (SEQ ID NO: 1 LCDR1, SEQ ID NO: 7 LCDR2, SEQ ID NO: 3 LCDR3, SEQ ID NO: 20 HCDR1, SEQ ID NO: 21 HCDR2, and SEQ ID NO: 10 HCDR3), any of An anti-CD40 antibody or an antigen-binding fragment of the anti-CD40 antibody. The anti-CD40 antibody according to claim 1, or an antigen-binding fragment of the anti-CD40 antibody, wherein the anti-CD40 antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

3. The antigen-binding fragment according to claim 1, wherein the anti-CD40 antibody or the antigen-binding fragment of the anti-CD40 antibody is Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab, diabody.

4. An isolated nucleic acid encoding the anti-CD40 antibody according to claim 1 or an antigen-binding fragment of the anti-CD40 antibody.

5. A vector comprising the nucleic acid according to claim 4, wherein the vector is an expression vector.

6. An immunoconjugate comprising the anti-CD40 antibody according to claim 1 or an antigen-binding fragment of the anti-CD40 antibody and a cytotoxic agent.

7. A pharmaceutical composition comprising the anti-CD40 antibody according to claim 1 or an antigen-binding fragment of the anti-CD40 antibody or the immunoconjugate according to claim 6, and a pharmaceutical adjuvant.

8. A pharmaceutical composition comprising the anti-CD40 antibody according to claim 1, or an antigen-binding fragment of said anti-CD40 antibody, or the immunoconjugate according to claim 6, a therapeutic agent, and a pharmaceutical adjuvant, wherein said therapeutic agent is selected from a chemotherapeutic agent, an anti-PD-1 antibody, an anti-PD-L1 antibody, and a cytotoxic agent.

9. A combination product comprising the anti-CD40 antibody according to claim 1, or an antigen-binding fragment of said anti-CD40 antibody, or the immunoconjugate according to claim 6, and one or more therapeutic agents which are a chemotherapeutic agent, a cytotoxic agent, an anti-PD-1 antibody or an anti-PD-L1 antibody.

10. Use of the anti-CD40 antibody according to claim 1, or an antigen-binding fragment of said anti-CD40 antibody, or the immunoconjugate according to claim 6 for the manufacture of a medicament for preventing or treating a tumor or an infectious disease in a subject.

11. Said tumor is at least any one of diffuse large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, metastatic breast cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma, multiple myeloma, chronic myeloid leukemia, stage III or stage IV melanoma which is a skin cancer, or Merkel cell carcinoma, head and neck squamous cell carcinoma (HNSCC), myelodysplastic syndrome, transitional cell carcinoma, pancreatic cancer, progressive or metastatic clear cell renal cell carcinoma which is clear cell renal cell carcinoma, and colon adenocarcinoma; said infectious disease is a bacterial infection, a viral infection, a fungal infection or a protozoal infection, and said infectious disease is a chronic infection. The use according to claim 10.

12. A kit for detecting CD40 in a sample, said kit comprising the anti-CD40 antibody according to claim 1, or an antigen-binding fragment of the anti-CD40 antibody. (a) contacting the sample with the anti-CD40 antibody according to claim 1, or an antigen-binding fragment of said anti-CD40 antibody; and (b) for performing the step of detecting the formation of a complex between said anti-CD40 antibody, or an antigen-binding fragment of said anti-CD40 antibody, and CD40, wherein said anti-CD40 antibody, or an antigen-binding fragment of said anti-CD40 antibody, is detectably labeled.

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