Antibody targeting human CD3 molecules and use thereof

Abstract

Provided is an antibody targeting human CD3 molecules. Compared with existing anti-CD3 antibodies, the provided antibody targeting human CD3 molecules has essentially the same activation effect or an even stronger activation effect on immune cells such as T cells, but at the same time triggers a smaller amount of cytokine release. The antibody targeting human CD3 molecules can further be used for preparing a bispecific antibody.

Description

Antibodies targeting human cd3 molecules and uses thereof

[0001] Cross-reference to related applications

[0002] This patent application claims the priority benefit of Chinese patent application No. CN202410828045.1, filed on June 25, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biological medicine, and specifically, the present application relates to an antibody or a functional fragment thereof targeting human CD3 molecules, and the application of the antibody or the functional fragment thereof. BACKGROUND

[0004] CD3 plays a key role in T cell activation and signal transduction. CD3 molecules are mainly distributed on the surface of mature T lymphocytes and are important differentiation antigens on the membrane of T cells. The subunits of CD3 molecules combine with the subunits of T cell receptors (TCR) to form heterodimers, which together constitute the TCR-CD3 complex. When TCR binds to antigen, CD3 target is activated, promoting downstream signal transduction, triggering the immune response of T cells, so that immune cells attack infected areas, tumors and other abnormal cells. Therefore, CD3 is a key regulatory point of T cell immune response, and intervention on CD3 target can regulate the degree of T cell activation and the intensity of immune response, which has important significance in the treatment of immune diseases and tumor immunotherapy.

[0005] CD3 protein has important clinical applications in the medical field. For example, CAR-T cell therapy in immunotherapy uses the knowledge of CD3 zeta signal to design artificial T cells that can more effectively recognize and attack tumor cells, and this treatment has achieved remarkable results in the treatment of some leukemia and lymphoma.

[0006] OKT3 is the first approved therapeutic CD3 antibody. OKT3 was first approved by the US FDA in 1985 for the treatment of acute rejection after organ transplantation, which is a mouse monoclonal antibody that can specifically react with the TCR-CD3 complex on the surface of T cells, and activates circulating T cells by binding to a glycoprotein CD3 epsilon chain. It is reported that OKT3 cannot bind to CD3-epsilon chain single transfected cells. That is, the recognition of OKT3 to CD3 epsilon chain is based on the conformational epitope on two or more CD3 subunits. This interaction leads to transient activation of T cells, release of cytokines, and prevents T cell proliferation and differentiation.

[0007] However, the use of anti-CD3 antibodies such as OKT3 also has certain problems. For example, the use of OKT3 is limited by the toxic dose response syndrome. This syndrome is believed to be associated with OKT3-mediated T cell activation and cytokine release. OKT3 is a potent mitogen that can promote T cell proliferation and cytokine secretion, triggering a series of side effects, including fever, chills, nausea, vomiting, and headache, summarized as "flu-like", "cytokine release" or "first dose" syndrome. A small number of patients experience more serious side effects such as cardiorespiratory distress, epilepsy, encephalopathy, meningitis, renal dysfunction, and graft thrombosis.

[0008] The long-term activation of primary T cells by anti-CD3 antibodies such as OKT3 can also lead to T cell anergy. T cell anergy is a tolerance mechanism in which lymphocytes are functionally inactivated after antigen encounter, but survive for a long time in a hypo-responsive state. T cell anergy is associated with persistent high-intensity activation of antigens in vitro or stimulation in an environment lacking costimulation or high co-inhibition.

[0009] Bispecific antibodies (BsAb), simply "dual antibodies", are a class of bifunctional antibody molecules that can specifically bind to two different antigens at the same time. Such molecules can bridge target cells (expressing one of the antigens, such as tumor cells expressing tumor-associated antigens) and functional molecules or their cells (such as T cells expressing T cell receptors), stimulate and guide the body's immune response, so as to better control and kill target cells, especially in tumor immunotherapy, which has broad application prospects.

[0010] Bispecific antibodies targeting CD3 as one of the target antigens are a class of dual antibodies that have been studied and applied more deeply. Bispecific antibodies with CD3 and tumor antigen binding activity can recruit T cells to tumors by binding TCR-CD3 complex, thereby bridging T cells and tumors and promoting T cell activation, and then killing tumors. There are many bispecific antibodies of different structural forms in the research, clinical trial or approved marketing stages. In addition to targeting CD3 as one of the target antigens, these bispecific antibodies also target CD19, CD20, CD123, CD33, CD38, B cell maturation antigen (BCMA) and other tumor-associated antigens.

[0011] However, the application of bispecific antibodies also has certain problems, the most important of which is that it can cause the circulating levels of inflammatory cytokines, including interferon-γ and interleukin-6, to increase, thereby causing cytotoxicity. This symptom of increasing the levels of cytokines in the whole body of a patient is also called a cytokine release storm (CRS). Cytokine release storm is a systemic inflammatory response, which is related to infectious diseases and non-infectious diseases, and can be induced by infection, drugs and other factors. CRS mainly affects all important organs of the human body, and can cause multiple organ failure in severe cases. In CRS, a large number of immune cells (B cells, T cells and / or natural killer cells, macrophages, dendritic cells and monocytes) are activated and then release more inflammatory cytokines. It has been reported that cytokine release usually begins within a few minutes to a few hours after infusion of antibodies such as CD28 superagonists and anti-CD3 (OKT3).

[0012] The degree of activation of immune cells determines the severity of CRS. In the application of bispecific antibodies taking CD3 as one of the targeted antigens, the activation of T cells depends on the role played by the activating CD3 antibody part.

[0013] In summary, there is still a need in the art for a new antibody targeting CD3, which should weaken the anergic state of T cells while reducing the cytokine release syndrome caused by activation; meanwhile, in the case of preparing a bispecific antibody using the new antibody, the bispecific antibody should cause immune cells to produce a smaller amount of cytokines while not triggering a high level of killing of tumor cells by T cells, thereby reducing the inflammatory response in vivo. SUMMARY

[0014] To solve the above technical problems, the purpose of the present application is to provide an antibody or antigen-binding fragment thereof targeting human CD3 molecules, which has substantially the same, or even stronger, activating effect on immune cells such as T cells (e.g., causing strong T cell proliferation, mediating significant tumor cell killing by T cells) compared to existing anti-CD3 antibodies, but at the same time causes the release of a smaller amount of cytokines, particularly inflammatory cytokines. The purpose of the present application is also to disclose the interaction mechanism of the antibody with human CD3 molecules based on the antibody provided by the present application, to explore the reasons why the antibody can achieve stronger T cell activation and cause the release of a smaller amount of cytokines, and to provide a new approach for developing better T cell activation reagents and immunotherapeutic drugs.

[0015] The present application provides the following technical solutions.

[0016] In one aspect, the present application provides an antibody or fragment thereof capable of specifically targeting or binding to CD3 on the surface of a human T cell, in particular the CD3 epsilon (e) subunit. Further, the present application provides an antibody or fragment thereof that binds only to the epsilon (e) subunit in the delta (d)-epsilon (e) heterodimer of CD3, but not to the epsilon (e) subunit in the gamma (g)-epsilon (e) heterodimer of CD3.

[0017] Depending on the context, the terms "targeting" and "binding" are used interchangeably in the present application.

[0018] In particular, the antibody or fragment thereof targeting CD3 molecules provided by the present application comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise a combination of complementarity determining regions (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of:

[0019] (i) CDRH1 comprising the amino acid sequence (GYTFSRY) shown in SEQ ID NO: 9, CDRH2 comprising the amino acid sequence (LPGSGS) shown in SEQ ID NO: 10, CDRH3 comprising the amino acid sequence (CYRYDAYGMDY) shown in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence (RASQSISGYLH) shown in SEQ ID NO: 13, CDRL2 comprising the amino acid sequence (YASQSIS) shown in SEQ ID NO: 14, CDRL3 comprising the amino acid sequence (QNGHSFPLT) shown in SEQ ID NO: 15;

[0020] (ii) CDRH1 comprising the amino acid sequence (GYTFSRYW) shown in SEQ ID NO: 19, CDRH2 comprising the amino acid sequence (ILPGSGST) shown in SEQ ID NO: 20, CDRH3 comprising the amino acid sequence (ARCYRYDAYGMDY) shown in SEQ ID NO: 21; and, CDRL1 comprising the amino acid sequence (QSISGY) shown in SEQ ID NO: 22, CDRL2 comprising the amino acid sequence (YAS) shown in SEQ ID NO: 23, CDRL3 comprising the amino acid sequence (QNGHSFPLT) shown in SEQ ID NO: 15;

[0021] (iii) CDRH1 containing the amino acid sequence (RYWIE) shown in SEQ ID NO:24, CDRH2 containing the amino acid sequence (EILPGSGSTNYNEKFKG) shown in SEQ ID NO:25, and CDRH3 containing the amino acid sequence (CYRYDAYGMDY) shown in SEQ ID NO:11; and CDRL1 containing the amino acid sequence (RASQSISGYLH) shown in SEQ ID NO:13, CDRL2 containing the amino acid sequence (YASQSIS) shown in SEQ ID NO:14, and CDRL3 containing the amino acid sequence (QNGHSFPLT) shown in SEQ ID NO:15; and

[0022] (iv) CDRH1 containing the amino acid sequence (GYTFSRY) shown in SEQ ID NO:9, CDRH2 containing the amino acid sequence (LPGSGS) shown in SEQ ID NO:10, CDRH3 containing the amino acid sequence (CYRYDAYGMDY) shown in SEQ ID NO:11; and CDRL1 containing the amino acid sequence (SGYLHWY) shown in SEQ ID NO:26, CDRL2 containing the amino acid sequence (LLIKYASQSI) shown in SEQ ID NO:27, and CDRL3 containing the amino acid sequence (QNGHSFPL) shown in SEQ ID NO:28.

[0023] In the context of this invention, the provided combination of light and heavy chain CDRs is derived from the heavy and light chain amino acid sequences of the antibodies specifically described in the embodiments of this invention, and those skilled in the art can conventionally determine the CDRs contained in the heavy and light chains of the antibody. For example, the combination of light and heavy chain CDRs provided above can be found in the "Best Mode for Carrying Out the Invention" section.

[0024] In the antibody or fragment thereof provided by the present invention, preferably, the antibody or fragment thereof targeting the CD3 molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0025] In the context of this invention, "at least 75% identity" means any percentage of identity between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0026] In the context of the present application, "heavy chain variable region" and "light chain variable region" both include the above-mentioned CDR combinations as well as the intervening framework regions, the arrangement of the individual domains being FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the "at least 75% identity" results in a difference of up to 25% in the amino acid sequence, which can be present in any of the framework regions of the heavy chain variable region or the light chain variable region, or in any of the domains or sequences outside the heavy chain variable region and the light chain variable region. The difference can be caused by deletion, addition or substitution of amino acids at any position.

[0027] In terms of the antigen, the antibody or fragment thereof of the present application is particularly an antibody or fragment thereof targeting a human CD3 molecule, in particular a human CD3 epsilon (e) subunit; preferably, the antibody is in any of the forms of a murine, chimeric, partially or fully humanized antibody, etc., or the antibody or fragment thereof targeting a CD3 molecule is a half-antibody or any antibody structure form capable of binding to a human CD3, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; more preferably, the antibody is an IgG. According to the specific embodiments of the present application, the present application provides an isolated and structurally characterized monoclonal antibody that specifically binds to CD3, in particular to CD3 on the surface of a human T cell.

[0028] In addition to the variable region, the antibody or fragment thereof provided by the present application further comprises a constant region, such as a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and / or a light chain constant region (CL); preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region.

[0029] According to the specific embodiments of the present application, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. Preferably, the monoclonal antibody comprises part or all of a human IgG1 subtype heavy chain constant region and a light chain constant region. For example, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 16; the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 1.

[0030] According to the embodiments of the present application, the antibody targeting CD3 epsilon molecule of the present application is a monoclonal antibody. Preferably, the antibody targeting CD3 epsilon molecule of the present application is an immunoglobulin. According to the embodiments of the present application, the antibody targeting CD3 epsilon molecule of the present application is an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 17, and a light chain having the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 18 (see the antibody designated as "4B1").

[0031] According to the requirements of practical applications, the antibody targeting CD3 epsilon molecule of the present application can be in the form of effector function null.

[0032] In another aspect, the present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof of the present application.

[0033] The nucleic acid molecule of the present application can be cloned into a vector, which in turn transforms or transfects a host cell. Therefore, in yet another aspect, the present application also provides a vector comprising the nucleic acid molecule of the present application. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a bacteriophage vector, etc. The vector or nucleic acid molecule of the present application can be used to transform or transfect a host cell. Therefore, in still another aspect, the present application provides a host cell comprising the nucleic acid molecule and / or vector of the present application, or a host cell transformed or transfected by the nucleic acid molecule and / or vector of the present application. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or an insect, fungal, plant or animal cell.

[0034] The antibody or fragment thereof of the present application can be obtained using any method known in the art. For example, the host cell of the present application is cultured under conditions allowing the expression of the heavy and light chains of the antibody. Optionally, the method further comprises the step of recovering the produced antibody.

[0035] The antibody or fragment thereof, nucleic acid molecule, vector and / or host cell of the present application can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical preparation, for various purposes according to the requirements. Therefore, in yet another aspect, the present application also provides a composition, such as a pharmaceutical composition, comprising the antibody or fragment thereof, nucleic acid molecule, vector and / or host cell of the present application, and optionally a pharmaceutically acceptable excipient.

[0036] Experiments have shown that the antibody or fragment thereof targeting CD3 molecules provided by the present application can effectively activate T cells, and after the heavy chain variable region and the light chain variable region contained therein are constructed into a bispecific antibody together with an antibody against other antigens, the T cells can still be effectively recruited at the tumor cell site, achieving good tumor cell killing effect, but at the same time, the inflammatory cytokines produced by T cells in response to the antibody or fragment thereof provided by the present application are relatively less in releasing inflammatory cytokines associated with cytokine storm. Therefore, the antibody or fragment thereof (e.g. functional region) targeting CD3 molecules provided by the present application can be used to construct a multispecific, in particular bispecific antibody, to play a role in binding the T cell surface CD3 to effectively recruit T cells and at the same time not to excessively stimulate T cells.

[0037] Therefore, in another aspect, the present application also provides the use of the antibody or fragment thereof targeting CD3 molecules, nucleic acid molecules, vectors, host cells and / or compositions in the preparation of a reagent for recruiting and activating T cells. The reagent can be a multispecific antibody, for example a bispecific antibody.

[0038] Preferably, the reagent is a bispecific antibody comprising the antibody or fragment thereof targeting CD3 molecules provided by the present application as a first binding domain. The first binding domain specifically binds to CD3 molecules and comprises a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the following:

[0039] (i) CDRH1 comprising the amino acid sequence (GYTFSRY) shown in SEQ ID NO: 9, CDRH2 comprising the amino acid sequence (LPGSGS) shown in SEQ ID NO: 10, CDRH3 comprising the amino acid sequence (CYRYDAYGMDY) shown in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence (RASQSISGYLH) shown in SEQ ID NO: 13, CDRL2 comprising the amino acid sequence (YASQSIS) shown in SEQ ID NO: 14, CDRL3 comprising the amino acid sequence (QNGHSFPLT) shown in SEQ ID NO: 15;

[0040] (ii) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 19 (GYTFSRYW), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 20 (ILPGSGST), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 21 (ARCYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 22 (QSISGY), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 23 (YAS), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (QNGHSFPLT);

[0041] (iii) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 24 (RYWIE), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 25 (EILPGSGSTNYNEKFKG), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (CYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (RASQSISGYLH), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (YASQSIS), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (QNGHSFPLT); and

[0042] (iv) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 9 (GYTFSRY), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 10 (LPGSGS), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (CYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 26 (SGYLHWY), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 27 (LIKYASQS I), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 28 (QNGHSFPL).

[0043] Further, the first binding domain specifically binds to a CD3 molecule and comprises a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence at least 75% identical to said amino acid sequence; and, a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence at least 75% identical to said amino acid sequence.

[0044] Further, the agent further comprises a second binding domain for specifically binding to a tumor associated antigen (TAA). For example, the bispecific antibody provided herein is capable of binding to a tumor associated antigen CEA, HER2 or CD19 via its second binding domain.

[0045] Accordingly, the present application also provides a multispecific antibody comprising: the antibody or fragment thereof targeting CD3 molecule as a first binding domain; and, a second binding domain capable of binding to a tumor associated antigen. As described above, the first binding domain specifically binds to CD3 molecule and comprises a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of:

[0046] (i) CDRH1 comprising the amino acid sequence (GYTFSRY) set forth in SEQ ID NO: 9, CDRH2 comprising the amino acid sequence (LPGSGS) set forth in SEQ ID NO: 10, CDRH3 comprising the amino acid sequence (CYRYDAYGMDY) set forth in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence (RASQSISGYLH) set forth in SEQ ID NO: 13, CDRL2 comprising the amino acid sequence (YASQSIS) set forth in SEQ ID NO: 14, CDRL3 comprising the amino acid sequence (QNGHSFPLT) set forth in SEQ ID NO: 15;

[0047] (ii) CDRH1 comprising the amino acid sequence (GYTFSRYW) set forth in SEQ ID NO: 19, CDRH2 comprising the amino acid sequence (ILPGSGST) set forth in SEQ ID NO: 20, CDRH3 comprising the amino acid sequence (ARCYRYDAYGMDY) set forth in SEQ ID NO: 21; and, CDRL1 comprising the amino acid sequence (QSISGY) set forth in SEQ ID NO: 22, CDRL2 comprising the amino acid sequence (YAS) set forth in SEQ ID NO: 23, CDRL3 comprising the amino acid sequence (QNGHSFPLT) set forth in SEQ ID NO: 15;

[0048] (iii) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 24 (RYWIE), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 25 (EILPGSGSTNYNEKFKG), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (CYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (RASQSISGYLH), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (YASQSIS), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (QNGHSFPLT); and

[0049] (iv) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 9 (GYTFSRY), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 10 (LPGSGS), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (CYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 26 (SGYLHWY), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 27 (LIKYASQSI), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 28 (QNGHSFPL).

[0050] Further, the first binding domain specifically binds to a CD3 molecule and comprises a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence at least 75% identical to said amino acid sequence; and, a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence at least 75% identical to said amino acid sequence.

[0051] According to the detailed description of the application, the multispecific antibody is a bispecific antibody. For example, the bispecific antibody provided by the application is capable of binding to a tumor-associated antigen CEA, HER2 or CD19 through its second binding domain.

[0052] The bispecific antibody provided by the application can take the form of a homodimer, i.e. having two identical heavy chains and two identical light chains, the heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC-anti-TAA scfv; the light chain comprising domains connected in the following order: anti-CD3 VL-CL.

[0053] Alternatively, the bispecific antibody provided by the present application can adopt a heterodimeric form, i.e. having two different heavy chains and two different light chains, one of the heavy chains comprising domains connected in the following order: anti-CD3 VH-CH1-FC, the other heavy chain comprising domains connected in the following order: anti-TAA VH-CH1-FC; one of the light chains comprising domains connected in the following order: anti-CD3 VL-CL, the other light chain comprising domains connected in the following order: anti-TAA VL-CL.

[0054] The bispecific antibody provided by the present application can be obtained by KiH technology, for example, using the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 as the heavy chain constant region of the two heavy chains of the bispecific antibody.

[0055] According to the specific embodiments of the present application, the tumor-associated antigen is CEA, HER2 or CD19. Accordingly, the second binding domain of the bispecific antibody provided by the present application specifically binds to CEA, CD19 or HER2.

[0056] Preferably, the second binding domain specifically binds to CEA and comprises a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of:

[0057] CDRH1 comprising the amino acid sequence (GFNIKDTY) shown in SEQ ID NO: 35, CDRH2 comprising the amino acid sequence (IDPANGNS) shown in SEQ ID NO: 36, CDRH3 comprising the amino acid sequence (APFGYYVSDYAMAY) shown in SEQ ID NO: 37; and, CDRL1 comprising the amino acid sequence (ESVDIFGVGF) shown in SEQ ID NO: 38, CDRL2 comprising the amino acid sequence (RAS) shown in SEQ ID NO: 39, CDRL3 comprising the amino acid sequence (QQTNEDPYT) shown in SEQ ID NO: 40.

[0058] Further, the second binding domain specifically binds to CEA and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to said amino acid sequence; and, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to said amino acid sequence.

[0059] Accordingly, the bispecific antibody can be referred to simply as "CD3 x CEA bispecific antibody". The CD3 x CEA bispecific antibody can take a homodimeric form, i.e. having two heavy chains and two light chains identical, the heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC-anti-CEA scfv; the light chain comprising domains connected in the following order: anti-CD3 VL-CL. The CD3 x CEA bispecific antibody can also take a heterodimeric form, i.e. having two heavy chains and two light chains different, one heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC, the other heavy chain comprising domains connected in the following order: anti-CEA VH-CH1-FC; one light chain comprising domains connected in the following order: anti-CD3 VL-CL, the other light chain comprising domains connected in the following order: anti-CEA VL-CL.

[0060] Alternatively, preferably, the second binding domain specifically binds to CD19 and comprises a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of:

[0061] CDRH1 comprising the amino acid sequence shown in SEQ ID NO: 41 (GYAFSSYW), CDRH2 comprising the amino acid sequence shown in SEQ ID NO: 42 (IWPGDSDT), CDRH3 comprising the amino acid sequence shown in SEQ ID NO: 43 (ARRETTTVGRYYYAMDY); and, CDRL1 comprising the amino acid sequence shown in SEQ ID NO: 44 (QSVDYSGDSY), CDRL2 comprising the amino acid sequence shown in SEQ ID NO: 45 (DAS), CDRL3 comprising the amino acid sequence shown in SEQ ID NO: 46 (QQSTENPWT).

[0062] Further, the second binding domain specifically binds to CD19 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 75% identity to said amino acid sequence; and, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to said amino acid sequence.

[0063] Accordingly, the bispecific antibody can be referred to simply as "CD3xCD19 bispecific antibody". The CD3xCD19 bispecific antibody can take a homodimeric form, i.e. having two heavy chains and two light chains identical, the heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC-anti-CD19 scfv; the light chain comprising domains connected in the following order: anti-CD3 VL-CL. The CD3xCD19 bispecific antibody can also take a heterodimeric form, i.e. having two heavy chains and two light chains different, one heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC, the other heavy chain comprising domains connected in the following order: anti-CD19 VH-CH1-FC; one light chain comprising domains connected in the following order: anti-CD3 VL-CL, the other light chain comprising domains connected in the following order: anti-CD19 VL-CL.

[0064] Alternatively, preferably, the second binding domain specifically binds HER2 and comprises a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of:

[0065] CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 47 (GYPFTQYF), CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 48 (ISSSYATV), CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 49 (VRSGNYEEYAMDY); and, CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 50 (QPLEYSNNQWNY), CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 51 (WAS), CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 52 (GQYSDYPNT).

[0066] Further, the second binding domain specifically binds HER2 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity to said amino acid sequence; and, the light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity to said amino acid sequence.

[0067] Accordingly, the bispecific antibody can be referred to as "CD3 x HER2 bispecific antibody" for short. The CD3 x HER2 bispecific antibody can take a homodimeric form, i.e. has two identical heavy chains and two identical light chains, the heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC-anti-HER2 scfv; the light chain comprising domains connected in the following order: anti-CD3 VL-CL. The CD3 x HER2 bispecific antibody can also take a heterodimeric form, i.e. has two different heavy chains and two different light chains, one of the heavy chains comprising domains connected in the following order: anti-CD3 VH-CH1-FC, the other heavy chain comprising domains connected in the following order: anti-HER2 VH-CH1-FC; one of the light chains comprising domains connected in the following order: anti-CD3 VL-CL, the other light chain comprising domains connected in the following order: anti-HER2 VL-CL.

[0068] By recruiting and triggering T cells to kill tumor cells, the antibody or fragment thereof targeting CD3 molecule provided by the present application or the reagent for recruiting and activating T cells prepared using the same can be used for immunotherapy, especially for the immunotherapy of tumors. Therefore, in another aspect, the present application also provides the use of the antibody or fragment thereof targeting CD3 molecule, nucleic acid molecule, vector, host cell, composition or the reagent for recruiting and activating T cells (such as a multispecific antibody) in the preparation of a medicament.

[0069] In clinic, the antibody against human CD3 molecule has been used for the treatment of acute rejection after organ transplantation, or for the activation and expansion of T cells, and further for cellular immunotherapy of solid tumors or hematological tumors. Therefore, the present application provides the use of the antibody or fragment thereof targeting CD3 molecule, nucleic acid molecule, vector, host cell, composition or the reagent for recruiting and activating T cells in the preparation of a medicament for the treatment of graft-versus-host disease, or for the treatment of tumors, such as solid tumors or hematological tumors, for example, gastric cancer, rectal cancer, breast cancer, kidney cancer, ovarian cancer, etc., for example, lymphoma, leukemia, myeloma, etc. The medicament prepared using the antibody targeting CD3 molecule of the present application can be a medicament with the antibody as the active ingredient, or a cell therapy medicament prepared using the antibody, such as CAR-T cells, TCR-T cells, etc.

[0070] In addition, the multispecific antibodies (e.g., bispecific antibodies) derived from anti-CD3 monoclonal antibodies are also currently used in clinic for treating tumors. According to the tumor-associated antigens (TAAs) that are highly expressed by tumor cells, a domain part targeting the TAAs is selected to be co-prepared into a bispecific antibody with a domain part targeting CD3 in the anti-CD3 antibody, so as to achieve the purpose of activating immune cells and enhancing immune cell-mediated tumor killing. Similarly, the present application also provides the use of the multispecific antibodies in the preparation of a medicament for treating graft-versus-host disease, or for treating tumors, such as solid tumors or hematological tumors, for example, gastric cancer, rectal cancer, breast cancer, kidney cancer, ovarian cancer, etc., or hematological tumors, for example, lymphoma, leukemia, myeloma, etc.

[0071] In another aspect, the present application also provides a method for treating graft-versus-host disease or tumors, which comprises administering the antibody or fragment thereof targeting CD3 molecules, nucleic acid molecules, vectors, host cells, compositions and / or multispecific antibodies of the present application to a subject in need. Preferably, the tumors are solid tumors or hematological tumors, for example, gastric cancer, rectal cancer, breast cancer, kidney cancer, ovarian cancer, etc., or hematological tumors, for example, lymphoma, leukemia, myeloma, etc. Among them, the subject is a mammal, more preferably a human.

[0072] The present application also provides a method for evaluating the efficacy of a T cell activating agent, which comprises contacting the T cell activating agent with a TCR-CD3 complex, and then detecting whether the agent binds only to the epsilon (ε) subunit in the delta (δ)-epsilon (ε) heterodimer of CD3, but not to the epsilon (ε) subunit in the gamma (γ)-epsilon (ε) heterodimer of CD3.

[0073] In the evaluation method, the TCR-CD3 complex can be a human TCR-CD3 complex.

[0074] Preferably, in the evaluation method, the T cell activating agent is an antibody or a fragment thereof, for example, a monoclonal antibody or a bispecific antibody or a fragment thereof. More preferably, the T cell activating agent is an antibody or a fragment thereof targeting CD3 molecules.

[0075] Preferably, in the evaluation method, the T cell activating agent is obtained by immunizing an animal with CD3, preferably human CD3, as an immunogen. Among them, the immunogen can also be a TCR-CD3 complex, preferably a human TCR-CD3 complex.

[0076] In the evaluation method, if it is determined that the T cell activating agent only binds to the epsilon (ε) subunit in the delta (δ)-epsilon (ε) heterodimer of CD3, but does not bind to the epsilon (ε) subunit in the gamma (γ)-epsilon (ε) heterodimer of CD3, it is determined that the T cell activating agent has good efficacy. For example, the agent will have a stronger activation effect on T cells but cause a smaller amount of inflammatory cytokine release compared to known anti-CD3 antibodies, such as OKT3 or UCHT1. The inflammatory cytokine can be an inflammatory cytokine associated with an early cytokine storm.

[0077] Preferably, in the evaluation method, the efficacy of the T cell activating agent refers to the efficacy of the agent in treating graft versus host disease or tumors. Further preferably, the tumor is a solid tumor or a hematological tumor. Preferably, the solid tumor is gastric cancer, rectal cancer, breast cancer, renal cancer or ovarian cancer; or, the hematological tumor is lymphoma, leukemia or myeloma.

[0078] Compared with the prior art, the present application provides a novel antibody targeting human CD3 molecules. Taking 4B1 provided in the embodiments of the present application as an example, experiments show that the antibody provided by the present application produces a very low level of inflammatory-related cytokines when activating human T cells, even less than one-third of the most widely used anti-CD3 antibody OKT3, but at the same time, the tumor killing effect produced by the antibody provided by the present application when activating human T cells is basically consistent with the level of OKT3. Therefore, the antibody targeting human CD3 molecules provided by the present application has greater application advantages in reducing side effects such as cytokine storm in the process of immunotherapy for tumor patients.

[0079] Further, the CD3 binding domain of the antibody targeting human CD3 molecule provided by the present application can also be used to construct bispecific antibodies. Therefore, the present application also provides bispecific antibodies prepared based on the novel antibody. As an example of bispecific antibodies constructed based on 4B1, experiments show that the CD3 binding domain of the monoclonal antibody 4B1 provided by the present application can be used to construct bispecific antibodies of different structures, which can mediate significant tumor cell killing effect. Compared with the bispecific antibodies using the CD3 binding domain of OKT3 as one of the targeting functional domains, the activation of T cells is stronger, and the amount of inflammatory cytokines related to cytokine storm is lower, which is the same as the effect of the monoclonal antibody 4B1. In vivo tumor killing experiments further show that the bispecific antibodies constructed based on the CD3 binding domain of the monoclonal antibody 4B1 provided by the present application have higher anti-tumor ability in vivo, and cause less inflammatory cytokines related to early cytokine storm. The results of solid tumor experiments show that the number of CD8+ T cells infiltrating the tumor of the mouse treated with the bispecific antibodies constructed based on the CD3 binding domain of the monoclonal antibody 4B1 provided by the present application is more, and the degree of exhaustion of CD8+ T cells is lower than that of the bispecific antibodies constructed based on the CD3 binding domain of OKT3. The low exhaustion degree of T cells leads to better tumor treatment effect.

[0080] The inventors further characterized the CD3 binding epitope of the antibody targeting human CD3 molecule of the present application. As an example of 4B1, experiments show that compared with OKT3 and UCHT1, the monoclonal antibody 4B1 targeting human CD3 molecule provided by the present application only binds to the ε subunit in the δ-ε heterodimer of CD3-TCR complex, but does not bind to the ε subunit in the γ-ε heterodimer. Without being limited by any theory, it is speculated that due to the binding to only one ε subunit in CD3, but not to two ε subunits (or to be precise, to only one ε subunit in the δ-ε heterodimer of CD3-TCR complex), the antibody of the present application can have a series of advantages compared with known anti-CD3 antibodies such as OKT3 and UCHT1, for example, as described above, can cause stronger T cell proliferation and mediate stronger tumor cell killing activity of T cells, and cause T cells to produce less inflammatory cytokines related to cytokine storm (thereby having less CRS effect). This finding provides an important basis for developing the next generation of antibodies targeting CD3 or TCR-CD3 complex. BRIEF DESCRIPTION OF DRAWINGS

[0081] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0082] Figure 1: Binding kinetics curve of monoclonal antibody to natural TCR-CD3 protein complex.

[0083] Figure 2: Detection of the activity of monoclonal antibodies in activating T cells to release cytokines.

[0084] Figure 3: Detection of the activity of monoclonal antibodies in causing T cell proliferation.

[0085] Figure 4: Schematic diagram of the structure of a monoclonal bispecific antibody.

[0086] Figure 5: Detection of the activity of bispecific antibodies in mediating PBMCs to kill tumor cells.

[0087] Figure 6: Detection of the activity of bispecific antibodies in mediating PBMCs to secrete cytokines.

[0088] Figure 7: Detection of the activity of bispecific antibodies in mediating PBMCs to kill tumor cells.

[0089] Figure 8: Pre-incubation of bispecific antibodies with T cells.

[0090] Figure 9: Detection of the activity of bispecific antibodies in mediating T cells to kill tumor cells and secrete cytokines after pre-incubation of bispecific antibodies with T cells.

[0091] Figure 10: Establishment of a mouse model and administration schedule for detection of the activity of monoclonal antibodies and bispecific antibodies in vivo.

[0092] Figure 11: Tumor progression curve for detection of activity in vivo.

[0093] Figure 12: In vivo tumor imaging for detection of activity in vivo.

[0094] Figure 13: Cytokine secretion in mice mediated by bispecific antibodies.

[0095] Figure 14: Effect of monoclonal antibodies and bispecific antibodies on the survival of mice.

[0096] Figure 15: Electron microscopy analysis of the binding of the Fab region of a monoclonal antibody to the TCR-CD3 protein complex.

[0097] Figure 16: Detection of the efficiency of the binding of a monoclonal antibody to human T cells.

[0098] Best mode for carrying out the invention

[0099] The present application is described below with reference to specific examples. Those skilled in the art will understand that these examples are for illustrative purposes only and do not limit the scope of the present application in any way.

[0100] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, and materials used in the following examples are commercially available unless otherwise specified. Among them:

[0101] The constant region sequence used in constructing chimeric antibodies is as follows:

[0102] Human IgG1 CL (SEQ ID NO:1):

[0103] Human IgG1 CH1 (SEQ ID NO:2):

[0104] Human IgG1 FC (SEQ ID NO:3):

[0105] When constructing bispecific antibodies, human IgG1 CL and the following constant region sequences containing Knob and Hole are used:

[0106] Heavy chain constant region (CH1 and FC-Knob) (SEQ ID NO:4):

[0107] Heavy chain constant region (CH1 and FC-Hole) (SEQ ID NO:5):

[0108] The preparation methods for chimeric monoclonal antibodies and bispecific antibodies are as follows:

[0109] Both chimeric monoclonal antibodies and bispecific antibodies were expressed in HEK-293F cells. Heavy chain and light chain plasmids were co-transfected in culture medium at a specific ratio. Transfected HEK-293F cells were cultured at 37°C for 3 days with shaking in a 5% CO2 incubator. The supernatant was centrifuged at 10,000 rpm for 5 min to obtain a clear expression supernatant. Antibody molecules were purified using protein A, and subsequent molecular sieving assessment showed high purity and a lack of aggregation (<1%) in the antibody protein.

[0110] The positive control antibodies (OKT3 and UCHT1) used in the examples were prepared in-house using known variable region sequences and the aforementioned constant region sequences.

[0111] This invention provides the sequence of antibody 4B1 (bold and underlined portions represent the signal peptide): 4B1:

[0112] Heavy chain (SEQ ID NO:6)

[0113] Light chain (SEQ ID NO:7)

[0114] The variable and constant regions of the heavy and light chains of antibody 4B1, as well as the full-length sequence, are shown below:

[0115] The CDR regions of antibody 4B1 were further obtained using different CDR definition schemes, as follows:

[0116] The anti-CEA (T48.6), anti-CD19 (B43), and anti-HER2 (HUA21) antibodies were used in the embodiments of the present application. The sequences of the light and heavy chain variable regions of the three antibodies are as follows, with the underlined bold portions being the CDR regions (partitioned in the IMGT way):

[0117] Anti-CEA antibody T48.6:

[0118] > heavy chain variable region (VH: SEQ ID NO: 29; CDRH1 / CDRH2 / CDRH3: SEQ ID NO: 35 / SEQ ID NO: 36 / SEQ ID NO: 37)

[0119] > light chain variable region (VL: SEQ ID NO: 30; CDRL1 / CDRL2 / CDRL3: SEQ ID NO: 38 / SEQ ID NO: 39 / SEQ ID NO: 40)

[0120] Anti-CD19 antibody B43:

[0121] > heavy chain variable region (VH: SEQ ID NO: 31; CDRH1 / CDRH2 / CDRH3: SEQ ID NO: 41 / SEQ ID NO: 42 / SEQ ID NO: 43)

[0122] > light chain variable region (VL: SEQ ID NO: 32; CDRL1 / CDRL2 / CDRL3: SEQ ID NO: 44 / SEQ ID NO: 45 / SEQ ID NO: 46)

[0123] Anti-HER2 antibody HUA21:

[0124] > heavy chain variable region (VH: SEQ ID NO: 33; CDRH1 / CDRH2 / CDRH3: SEQ ID NO: 47 / SEQ ID NO: 48 / SEQ ID NO: 49)

[0125] Light chain variable region (VL: SEQ ID NO: 34; CDRL1 / CDRL2 / CDRL3: SEQ ID NO: 50 / SEQ ID NO: 51 / SEQ ID NO: 52)

[0126] Example 1 Preparation and screening of monoclonal antibodies

[0127] Two Bal b / c mice were immunized with human full-length TCR-CD3 complex (PDB database sequence number: 6JXR (Structure of human T cell receptor-CD3 complex)) and the serum titer was determined by indirect ELISA.

[0128] The human CD3 antigen was coated on a solid carrier, and a sample containing the mouse serum antibody to be tested was added to bind to the solid-phase antigen (Ag-Ab), and then an enzyme-labeled anti-CD3 antibody (secondary antibody) was added to bind to the above Ag-Ab complex to form a complex. Substrate was added, and the enzyme on the complex catalyzed the substrate to develop color. The degree of color development of the enzyme and the substrate was used to determine the titer of the antibody to be tested. There was a washing step between each step. If the sample does not contain the corresponding antibody, the enzyme-labeled antibody is washed away, the substrate does not develop color and shows a negative reaction. If the OD of the test well is greater than 0.1 and greater than 2.1 times the OD of the negative control well (P / N>2.1, where P is the OD value of the test serum at a certain dilution, and N is the OD value of the negative serum at the corresponding dilution), it is determined to be positive. The highest dilution of serum that is determined to be positive is the serum antibody titer. Monoclonal antibodies with a titer greater than 1:100K are selected for further functional experiments.

[0129] Using the feature that the surface BCR of memory B cells (MBC) specifically binds to the antigen and the surface markers of B cells, antigen-specific MBCs were sorted from the prepared PBMCs by flow cytometry. The sorted antigen-specific MBCs were obtained by extracting total RNA, RT-PCR amplification, vector construction, high-throughput expression to obtain cell supernatant containing monoclonal antibodies, and then detected by indirect ELISA. A certain number of positive clone cells were selected, the variable region sequences of the antibody light and heavy chains in the positive clone cells were measured, and the commercially available human IgG1 constant region vector was used to construct a heavy and light chain expression vector for expression and purification, and finally monoclonal antibodies were obtained.

[0130] Several murine monoclonal antibodies were obtained, designated as 4B1, 4B12, 4G3, 4D10, and 4F7.

[0131] Example 2 Affinity determination of monoclonal antibodies

[0132] The binding affinity of the monoclonal antibody and the positive control antibody to the natural TCR-CD3 protein complex is determined by a bio-layer interferometry method, and the equilibrium dissociation constant (KD) value is calculated.

[0133] Bio-layer interferometry assay was performed on an Octet RED96 system (ForteBio) at 30°C with 25 mM HEPES (pH 7.5), 150 mM NaCl and 0.06% glycyrrhizin saponin TCR-CD3 as the buffer. The TCR-CD3 protein was biotinylated with NHS-LC-LC biotin, and then loaded onto a streptavidin biosensor (ForteBio), which was equilibrated in the running buffer for 10 minutes. After 2 minutes of incubation, the biosensor was re-acted with different concentrations of monoclonal antibodies for 2 minutes, and then dissociated for 3 minutes. The data were analyzed using the global fitting algorithm in Octet data analysis software v.9.0 (ForteBio).

[0134] The results are shown in Figure 1.

[0135] The results of three independent experiments showed that for the target protein, 4B1 and 4F7 exhibited similar high affinity (pM level) as UCHT1, while OKT3 exhibited lower affinity (0.6 nM). The remaining antibodies 4G3 (1.6 nM), 4B12 (144 nM) and 4D10 (1800 nM) exhibited different degrees of affinity.

[0136] Example 3 Detection of the activity of monoclonal antibodies in activating T cells to release cytokines

[0137] Human primary T cells were purified from PBMC of healthy donors using human CD3 negative selection kit. Different concentrations of monoclonal antibodies were pre-coated on 96-well plates overnight at 4°C, and 1e5 human primary T cells were added to each well of the plate.

[0138] CD69: At 24h, the cultured T cells were taken, and the ratio of CD69 positive cells was detected by flow cytometry.

[0139] Culture supernatant cytokines: At 48h, the culture supernatant was taken, and detected by LEGEND-plex Human Thelper 1 (Th1) Cytokine Kit (containing IL-6, IFN-γ and TNF-α), and analyzed by flow cytometry according to the kit instructions.

[0140] Intracellular cytokines: T cells were taken at 48h, surface stained, fixed with 1% paraformaldehyde (4°C for 30min), permeabilized with 0.2% tween 20 (37°C for 20min), and stained intracellularly for perforin and GZMB. The percentage of T cells expressing perforin and GZMB was determined by flow cytometry.

[0141] Results are shown in Figure 2.

[0142] The results show that of the five monoclonal antibodies, 4B1 and UCHT1 activated T cells most efficiently after 24h co-culture (2A in Figure 2), with 9% more CD69 expression than OKT3, followed by 4B12, 4F7, 4G3 and 4D10. After 48h co-culture (2B in Figure 2), 4B1 activated T cells produced significantly more cytotoxic cytokines GZMB and Perforin than the other selected monoclonal antibodies, and at the same level as OKT3.

[0143] However, 4B1 mediated less production of inflammatory cytokines than OKT3 in terms of the inflammatory cytokine storm associated with T cell response to monoclonal antibody activation; and UCHT1 activated T cells produced very strong cytokine levels, even higher than OKT3.

[0144] Based on the above results, it can be preliminarily inferred that, compared with OKT3, monoclonal antibody 4B1 can produce lower levels of cytokines on the basis of causing high levels of T cell killing.

[0145] Example 4 Detection of the activity of monoclonal antibodies in causing T cell proliferation

[0146] The Leu234 and Leu235 mutations to alanine and the Pro329 mutation to glycine were introduced into 4B1 and OKT3, respectively, to prepare Fc silent variants of both, which remove the FC-mediated complement effect.

[0147] Human primary T cells were purified from PBMC of healthy donors using a human CD3 negative selection kit. Different concentrations of the above monoclonal antibody variants were pre-coated on 96-well plates overnight at 4°C, and 1e5 human primary T cells were added to each well of the plate, together with 3μg / mL anti-huCD28. After 96h incubation with the antibodies, T cells were taken and the percentage of exhausted PD-1 on CD4+ and CD8+ T cells was determined by flow cytometry.

[0148] Results are shown in Figure 3.

[0149] The experimental results show that 4B1 can cause stronger T cell proliferation; and after 4 days of culture, the expression level of cell surface exhaustion indicator PD-1 caused by 4B1 is lower than that of OKT3 stimulation, whether CD4+ or CD8+ T cells.

[0150] Example 5 Construction of bispecific antibodies (BsAb)

[0151] The monoclonal antibody 4B1 and the control antibody OKT3 provided by the present application are used to construct two structural forms of bispecific antibodies. The structure of the bispecific antibody is shown in Figure 4, wherein the bispecific antibody with the structure shown on the left side of Figure 4 is named CD3 / TAA (i.e. "IgG-scFv format"), and the bispecific antibody with the structure shown on the right side of Figure 4 is named CD3 x TAA (i.e. "knobs-into-holes format"), and TAA is respectively carcinoembryonic antigen CEA, HER2 and CD19.

[0152] First, the light and heavy chain variable region sequences of anti-CEA (T48.6), anti-CD19 (B43), and anti-HER2 (HUA21) antibodies are obtained from the Protein Data Bank of the United States.

[0153] For the CD3 / TAA format antibody, the heavy chain C-terminus of OKT3 and 4B1 monoclonal antibody variants (i.e. Fc silence) is connected to the scfv form of anti-TAA through a (GGGGS)1 linker, and a (GGGGS)4 long linker is used to connect the light and heavy chain variable regions of the scfv to construct a heavy chain of "anti-CD3 VH-CH1-FC-anti-TAA scfv". The light chain of OKT3 and 4B1 monoclonal antibodies is obtained. As described above, the heavy chain plasmid and the light chain plasmid are co-transfected into cells at a ratio of 1:1 to obtain bispecific antibodies of the CD3 / TAA format, which are named OKT3 / TAA and 4B1 / TAA, respectively.

[0154] For the CD3 x TAA format antibody, the heavy chain variable region of OKT3 and 4B1 monoclonal antibody is used to construct a heavy chain with the heavy chain constant region (CH1 and FC, containing hole mutation), and the heavy chain variable region of the above anti-TAA is used to construct a heavy chain with the heavy chain constant region (CH1 and FC, containing knob mutation). The light chain of OKT3 and 4B1 monoclonal antibodies is obtained. Similarly, the light chain of human IgG1 of anti-TAA is constructed. As described above, the two heavy chain plasmids and the light chain plasmid are co-transfected into cells at a ratio of 1:1:1 to obtain bispecific antibodies of the CD3 x TAA format, which are named OKT3 x TAA and 4B1 x TAA, respectively.

[0155] Example 6 Detection of the activity of bispecific antibodies in mediating PBMCs to kill tumor cells and secrete cytokines

[0156] (1) Targeting CEA

[0157] Human gastric cancer cell line MKN45 expressing CEA antigen was purchased from National Cell Line Resource (Beijing, China) and cultured in 1640 medium (Gibco) supplemented with 1% L-glutamine, 1% penicillin / streptomycin and 10% fetal bovine serum.

[0158] Human MKN45 cells were mixed with in vitro isolated human CD3+T cells at a ratio of 10:1 E:T. Cells were incubated with limited dilution OKT3 BsAb, 4B1 BsAb or control BsAb that does not bind to the specific tumor cell line. After 48 hours incubation at 37°C, lactate dehydrogenase (LDH) release assay was performed using LDH release kit (Bi Yun Tian) according to the manufacturer's instruction.

[0159] The results are shown in Figures 5 and 6.

[0160] The experimental results show that both BsAbs against carcinoembryonic antigen (CEA) have high activity in killing human MKN45 gastric cancer cell line (high expression of CEA antigen) in PBMCs prepared from healthy donors. Among them, Figure 5 shows the representative dose-response curves of 9 healthy donors, as well as the summary of EC50 values and killing degree, indicating that the average EC50 of CD3_4B1 / CEA induced antibody killing tumor is higher than that of CD3_OKT3 / CEA, but there is no difference in the average killing degree between the two; Figure 6 shows the results of cytokine levels, i.e. interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the supernatant of culture fluid, indicating that the cytokine release stimulated by CD3_4B1 / CEA is significantly lower than that of CD3_OKT3 / CEA group.

[0161] (2) Targeting CEA, HER2 or CD19

[0162] Further in vitro detection of the killing activity of the bispecific antibodies constructed in the present application on carcinoembryonic antigen expressing cells, HER2 expressing tumor cells (SKOV3 cells, as an example of solid malignant tumor), CD19 expressing tumor cells (RAJI cells, as an example of hematological malignancies).

[0163] Human tumor cell lines expressing specific antigens were mixed with in vitro isolated human CD3+ T cells at 10:1 E:T ratio. 96-well plates contained 3e4 tumor cells and 3e5 human primary T cells per well. Cells were incubated with a concentration gradient of OKT3 BsAb, 4B1 BsAb. After 24 (RAJI) or 48 (SKOV3) hours of incubation at 37°C, lactate dehydrogenase (LDH) release assay was performed using LDH lactate dehydrogenase release kit according to the manufacturer's instructions.

[0164] Results are shown in Figure 7.

[0165] The results of the three tumor killing models showed that the average EC50 of antibody killing tumor caused by CD3_4B1 / TAA was higher than that of CD3_OKT3 / TAA, but there was no difference in the average killing percentage of the two.

[0166] Example 7 Detection of the activity of bispecific antibodies in mediating T cell killing of tumor cells and secretion of cytokines after pre-incubation of T cells with bispecific antibodies

[0167] Because the BsAb mainly acts on T cells, in order to exclude the influence of other cells in PBMCs, a treatment method of pre-incubation of bispecific antibodies with T cells was established, as shown in Figure 8.

[0168] As shown in the figure, after pre-incubation of T cells with BsAb for 25 min (flow identification showed that all antibodies had been bound to T cells) and washing steps, the target cells, i.e. tumor cells, were co-cultured, and then the killing percentage of tumor cells and Th1 cytokines (IL-2, IL-6, IL-10, IFN-γ and TNF-α) in the supernatant were detected; other conditions refer to the detection method used in Example 6 when PBMCs were used.

[0169] Results are shown in Figure 9.

[0170] The experimental results showed that, compared with PBMCs-BsAb+tumor cells, IL-6, IL-10 and TNF-α produced in the case of pre-incubation of T cells were significantly reduced; and after pre-incubation, the ratio of the maximum cytokine release of 4B1 / OKT3 was significantly lower than that of the PBMCs-BsAb+tumor cell group, while the killing percentage of tumor cells of the two groups was basically flat. It is suggested that the 4B1 antibody will achieve the effect of reducing the side effects of cytokine storm in the patient's body when used to construct bispecific antibodies for killing tumors in the patient's body, while it will not affect the killing ability of bispecific antibodies to tumors.

[0171] Example 8 Detection of the in vivo anti-tumor, cytokine release stimulating activity of bispecific antibodies and the effect on animal survival

[0172] To confirm that the in vitro killing and cytokine release profile of the bispecific antibodies containing CD3_4B1 are consistent with the in vivo efficacy, the RAJI lymphoblastoid lymphoma xenograft mouse model was used to compare the effector function of CD3_4B1xCD19 and CD3_OKT3xCD19.

[0173] The mouse model was established and the dosing regimen is shown in Figure 10. As shown in Figure 10, 5e5 RAJI lymphoblastoid lymphoma cells stably expressing luciferase (Lucifrase, LUC) were injected into the tail vein of the mice on day 0, and the mice were monitored for tumor progression. After confirming that the tumor progression was as expected, the pre-incubated T cells and PBMCs were injected into the tail vein of the mice on day 7. 5e6 T cells were pre-incubated with different concentration gradients of the bispecific antibodies for 30 minutes at a concentration of 1e7 / ml, and the excess bispecific antibodies were removed by PBS washing twice. The pre-incubated T cells were mixed with 1e7 PBMCs and injected into the mice through the tail vein, and then the bispecific antibodies constructed in the application and 4B1 or OKT3 monoclonal antibodies were injected into the tail vein of the mice at a dose of 30, 10, 2, and 0.2 μg per mouse on days 10, 14, 18, and 22, respectively.

[0174] (1) Tumor progression

[0175] The mice were evaluated for tumor burden using bioluminescence imaging before each antibody administration and on days 7-22. Each mouse was intraperitoneally injected with 10 mg / ml d-luc luciferin substrate, and after 10 minutes of anesthesia with isoflurane, the light intensity change was detected by bioluminescence imaging technology (BLI) to monitor the disease development in real time.

[0176] The results are shown in Figures 11 and 12.

[0177] The experimental results show that the CD3_4B1xCD19 molecule can produce strong tumor cell killing at various dose levels, and the 10 μg dose shows almost complete tumor clearance, and the 30 μg dose is similar to the 10 μg dose, but shows a higher level of inflammatory factors, which indicates that the 10 μg dose has the best therapeutic effect on the mice under the premise of the least cytokines. Moreover, the bioluminescence photos show that CD3_4B1xCD19 effectively controls the growth of RAJI tumor cells, and the mice maintain a very low tumor level within 20 days after treatment. Notably, the tumor burden of CD3_4B1xCD19 at a dose of 10 μg is more obvious than that of CD3_OKT3xCD19 in terms of tumor clearance.

[0178] (2) Cytokine secretion

[0179] It is known from the study of clinical patients treated with BsAb immunotherapy complicated by cytokine storm that the level of cytokines usually reaches a peak after the first immunotherapy, causing irreversible damage to the patient's whole body, including the cardiovascular and nervous systems.

[0180] To study the cytokine levels in mice treated with two BsAbs, mouse peripheral blood serum was taken at different time points (2, 6, and 24 hours) within 24 hours after the first BsAb treatment: tumor progression on the 7th day. After centrifugation of about 100 ul of mouse serum at room temperature at 3000 rpm for 10 minutes, the supernatant was taken. Detection was performed by LEGEND-plex Human T helper 1 (Th1) Cytokine Kit (containing IL-10, IL-2, IL-6, IFN-γ, and TNF-α), and flow cytometry analysis was performed according to the kit instructions. The secretion of human Th1-type cytokines was detected.

[0181] The results are shown in Figure 13.

[0182] The experimental results show that the five cytokines have similar metabolic peak degrees, with a peak of secretion at 2 hours, a gradual decrease at 6 hours, and a return to baseline within 24 hours. Consistent with previous in vitro results, the cytokines produced by CD3_4B1 x CD19-activated immune systems were significantly lower than those produced by CD3_OKT3 x CD19. This indicates that the bispecific antibody based on CD3_4B1 can effectively reduce the cytokine storm that occurs in a short period of time, achieving the purpose of optimizing treatment.

[0183] (3) Animal survival

[0184] It is well known that RAJI lymphoma has strong proliferation and invasion ability in vivo, which can greatly threaten the survival of NKG mice in a short period of time. Therefore, the effect of treatment with two immunotherapeutic bispecific antibodies on the survival of tumor-bearing mice was detected.

[0185] From the 7th to the 35th day of the above experiment, the survival of mice was recorded by daily observation, and the Kaplan-Meier mouse survival curve was plotted (PRISM 8.0 software).

[0186] The results are shown in Figure 14.

[0187] From the results of the mouse survival curve, it can be seen that the mice in the control and vehicle groups basically died within 25 days of tumor inoculation. After treatment with CD3_4B1 x CD19 and CD3_OKT3 x CD19 formulations, the mice basically survived within 35 days.

[0188] Example 9 Epitope characterization of monoclonal antibody binding to CD3

[0189] As shown in the above examples, the anti-CD3 antibody 4B1 and bispecific antibodies constructed using 4B1 provided by the present application can cause stronger T cell proliferation and can mediate stronger tumor cell killing activity of T cells, but at the same time cause T cells to produce less inflammatory cytokines related to cytokine storm, relative to the control antibody OKT3 and bispecific antibodies constructed using OKT3. The following experiments were performed to further study the mechanism of action of 4B1 and its functional domains.

[0190] Recombinant vectors for expressing light chains and heavy chains of anti-CD3 antibodies OKT3, UCHT1, and 4B1 were constructed using pcDNA3.4, and then HEK293 free style cells were co-transfected, recombinant antibodies were expressed, supernatants were collected, and anti-CD3 antibodies OKT3, UCHT1, and 4B1 were purified. Then, Fab was obtained by papain digestion. The TCR-CD3 complex was incubated with the Fab of the three antibodies at a 1:1.5 molar ratio at 4°C for 3 h, and then gel column filtration was performed to obtain 4B1-TCR-CD3, OKT3-TCR-CD3, and UCHT1-TCR-CD3 protein complexes.

[0191] The 4B1-TCR-CD3 protein complex was concentrated for cryo-EM analysis and image reconstruction, and a cryo-EM structure image with a global resolution of 3.2 A was obtained, as shown in Fig. 15A. As shown in Fig. 15A, the Fab molecules of 4B1 form a 1:1 stoichiometric complex with the TCR-CD3 complex, and the Fab molecules of 4B1 bind to the CD3δ-ε heterodimer. At the same time, the cryo-EM structure images of OKT3-TCR-CD3 and UCHT1-TCR-CD3 complexes were determined at resolutions of 3.4 A and 3.3 A, respectively, as shown in Figs. 15B and 15C. As shown in the figures, the Fab molecules of OKT3 and UCHT1 both form a 2:1 stoichiometric complex with the TCR-CD3 complex, compared with the 1:1 stoichiometry between 4B1 Fab and TCR-CD3.

[0192] ​​​Analysis of the binding epitopes of OKT3, UCHT1, 4B1 on CD3 shows that the Fab molecules of 4B1, OKT3 and UCHT1 recognize the overlapping epitopes on the CD3e subunit of CD3d-e heterodimer, but their interactions with CD3e are significantly different: there is an angle deviation of about 45° of 4B1 Fab relative to OKT3 Fab and UCHT1 Fab, so as to interact with CD3e of CD3d-e heterodimer, see 15D in Figure 15; if 4B1 Fab is docked with the CD3e subunit in CD3g-e heterodimer, there will be serious spatial conflict with the TCR b subunit, see 15E in Figure 15. These observations confirm the 1:1 stoichiometry between the Fab molecules of 4B1 and the TCR-CD3 complex.

[0193] Further, the binding efficiency of 4B1, OKT3 and UCHT1 antibodies to human T cells was detected by flow cytometry.

[0194] Human CD3+T cells were collected, washed with PBS, and 5x10 5 The CD3+T cells were incubated with different concentrations of antibodies at 4°C for 30 minutes; anti-human IgG antibody was used as isotype control. After incubation, the cells were washed with PBS, anti-human IgG-FITC antibody was added and incubated at 4°C for 30 minutes in the dark, and then washed again. The FITC fluorescence intensity on the cell surface was acquired, and analyzed by FlowJo software.

[0195] The results show that both antibodies exhibit effective binding to the TCR-CD3 complex. However, the mean fluorescence intensity (MFI) analysis shows that the amount of antibody bound on a single CD3+T cell by OKT3 at saturating concentration is twice the amount bound by 4B1; see Figure 16. In combination with the above electron microscopy structure analysis, it is further confirmed that OKT3 binds to the CD3e subunit of CD3d-e heterodimer and the CD3e subunit of CD3g-e heterodimer, while the anti-CD3 antibody 4B1 of the present application only binds to the CD3e subunit of CD3d-e heterodimer.

[0196] The above description of specific embodiments of the present application is not intended to limit the present application, and those skilled in the art can make various changes or modifications to the present application without departing from the spirit of the present application, and all such changes or modifications shall fall within the scope of the appended claims of the present application.

Claims

1. An antibody or fragment thereof targeting a CD3 molecule, characterized in that, The antibody or fragment thereof binds only to the epsilon (e) subunit in the delta (d)-epsilon (e) heterodimer of the CD3 molecule, and not to the epsilon (e) subunit in the gamma (g)-epsilon (e) heterodimer of the CD3 molecule.

2. The antibody or fragment thereof of claim 1, wherein, The antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain and light chain comprise a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of: (i) CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 9, CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 10, CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 14, CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (ii) CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 19, CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 20, CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 21; and, CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 22, CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 23, CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (iii) CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 24, CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 14, CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and (iv) CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 9, CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 10, CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 26, CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 27, CDRL3 comprising the amino acid sequence set forth in SEQ ID NO:

28.

3. The antibody or fragment thereof of claim 1 or 2, wherein, the antibody or fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to said amino acid sequence; and / or, a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to said amino acid sequence; Preferably, the antibody or fragment thereof is an antibody or antigen-binding fragment thereof targeting a human CD3 molecule; Preferably, the antibody is in any of the forms of a murine, chimeric, partially or fully humanized antibody, etc., or the antibody or fragment thereof targeting a human CD3 molecule is in any antibody structural form capable of binding to a human CD3, such as a scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

4. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof of any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 and / or the vector of claim 5.

7. A composition comprising the antibody or fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, and / or the host cell of claim 6; Preferably, the composition is a pharmaceutical composition, further comprising optional pharmaceutically acceptable excipients.

8. Use of the antibody or fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, and / or the composition of claim 7 for the manufacture of a reagent for recruiting and activating T cells; Preferably, the reagent is a multispecific antibody, preferably a bispecific antibody.

9. A multispecific antibody comprising the antibody or fragment thereof of any one of claims 1 to 3 as a first binding domain; and, the multispecific antibody further comprises a second binding domain capable of binding to a tumor antigen; Preferably, the multispecific antibody is a bispecific antibody.

10. The multispecific antibody of claim 9, wherein the multispecific antibody is a bispecific antibody; Preferably, the bispecific antibody is in a homodimeric or heterodimeric form.

11. Use of the antibody or fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, the composition of claim 7, and / or the multispecific antibody of claim 9 or 10 for the manufacture of a medicament for the treatment of graft versus host disease, or for the treatment of a tumor.

12. A method of treating graft versus host disease or a tumor, the method comprising administering to a subject in need thereof the antibody or fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, the composition of claim 7, and / or the multispecific antibody of claim 9 or 10.

13. Use according to claim 11 or method according to claim 12, characterized in that, the tumor is a solid tumor or a hematological tumor; preferably, the solid tumor is gastric cancer, rectal cancer, breast cancer, renal cancer or ovarian cancer; or, the hematological tumor is lymphoma, leukemia or myeloma; preferably, the subject is a mammal, preferably a human.

14. A method for evaluating the efficacy of a T-cell activating agent, characterized in that, the evaluation method comprises contacting a T cell activating agent with a TCR-CD3 complex, and then detecting whether the agent binds only to the epsilon (ε) subunit of the delta (δ)-epsilon (ε) heterodimer of CD3, but not to the epsilon (ε) subunit of the gamma (γ)-epsilon (ε) heterodimer of CD3.

15. The method of evaluating the efficacy of a drug according to claim 14, wherein the T cell activating agent is an antibody or fragment thereof, such as a monoclonal antibody or a bispecific antibody or fragment thereof; preferably, the T cell activating agent is an antibody or fragment thereof targeting a CD3 molecule; preferably, the TCR-CD3 complex is a human TCR-CD3 complex.

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