Ctla-4 binding molecules and uses thereof

Abstract

The present invention relates to specific CTLA-4 binding molecules and immunoconjugates, compositions containing the CTLA-4 binding molecules. The present invention also relates to nucleic acids encoding the CTLA-4 binding molecules and host cells comprising the same, as well as methods of making the CTLA-4 binding molecules. Furthermore, the present invention relates to therapeutic and diagnostic uses of these CTLA-4 binding molecules. In particular, the present invention relates to the combination treatment of these CTLA-4 binding molecules with other therapies, e.g. therapeutic modalities or therapeutic agents.

Description

[0001] This application is a divisional application of Chinese patent application 202010047300.0, the original application being filed on January 16, 2020, and entitled "CTLA-4 binding molecule and its use". Technical Field

[0002] This invention relates to specific CTLA-4 binding molecules and immunoconjugates and compositions containing said CTLA-4 binding molecules. Furthermore, this invention relates to nucleic acids encoding said CTLA-4 binding molecules and host cells containing them, as well as methods for preparing said CTLA-4 binding molecules. This invention also relates to the therapeutic and diagnostic uses of these CTLA-4 binding molecules, and in particular, to the combination therapy of these CTLA-4 binding molecules with other therapies, such as modes of treatment or therapeutic agents. Background Technology

[0003] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, is a leukocyte differentiation antigen. As a transmembrane protein belonging to the immunoglobulin superfamily, it is primarily expressed on the surface of activated T lymphocytes and B cells.

[0004] CTLA-4, expressed on the surface of T cells, shares high homology with the co-stimulatory molecule CD28 on the T cell surface, possessing the same ligands CD80 (B7-1) or CD86 (B7-2). However, CTLA-4 binds to both CD80 and CD86 with greater affinity and co-existence than CD28. The CTLA-4 / CD80 and / or CTLA-4 / CD86 complexes formed after CTLA-4 binds to CD80 (B7-1) or CD86 (B7-2) negatively regulate T cell receptor signaling, leading to subsequent downregulation of T cell activation, suppression of anti-tumor immune activity, and tumor immune escape. CTLA-4 is overexpressed in many cancer tissues, including various solid tumors such as melanoma, renal cell carcinoma, non-small cell lung cancer, liver cancer, and breast cancer. Overexpression of CTLA-4 on the surface of activated T lymphocytes promotes tumor invasion and is often associated with poor prognosis.

[0005] The successful application of monoclonal antibodies in cancer detection and targeted therapy has revolutionized tumor treatment. However, traditional monoclonal antibodies (150kD) have excessively large molecular weights, making it difficult to penetrate tissues and resulting in low effective concentrations in tumor areas and insufficient therapeutic effects. Furthermore, the long development cycle, high production costs, and insufficient stability of fully humanized traditional antibodies limit their clinical application and widespread use. Therefore, there is a need to develop new antibody molecules with smaller molecular weights.

[0006] Single-domain antibodies (sdAbs) are currently the smallest antibody molecules, with a molecular weight only 1 / 10 that of ordinary antibodies. In addition to possessing the antigenic reactivity of monoclonal antibodies, sdAbs also have some unique functional characteristics, such as small molecular weight, high stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, and low preparation cost. They almost perfectly overcome the shortcomings of traditional antibodies, such as long development cycles, low stability, and stringent storage conditions.

[0007] Currently, there are few single-domain antibody drugs targeting CTLA-4 in existing technologies, and the therapeutic effects of these single-domain antibody drugs are not ideal. Therefore, there is an urgent need in the field to develop new CTLA-4 binding molecules containing single-domain antibodies that effectively target CTLA-4. Summary of the Invention

[0008] Through dedicated research, the inventors have developed a group of CTLA-4 binding molecules containing a single-domain antibody (sdAb) moiety that specifically recognizes CTLA-4, which can...

[0009] (1) Binding CTLA-4 with high affinity, such as human CTLA-4, for example, the Kd of the binding between the CTLA-4 binding molecule and CTLA-4 is about 10. -5 M to approximately 10 -12 M, preferably, about 10 -7 M to approximately 10 -12 M;

[0010] (2) Effectively block the related activities of CTLA-4, for example, effectively block the binding of CTLA-4 to CD80 and / or CD86;

[0011] (3) Effectively induces the secretion of IL-2 and TNF-α; and

[0012] (4) Keep T cells in an activated state.

[0013] Therefore, in a first aspect, the present invention provides a CTLA-4 binding molecule comprising at least one CTLA-4-specific single-domain antibody (sdAb) moiety, said sdAb moiety comprising three complementarity-determining regions, namely CDR1, CDR2, and CDR3, wherein:

[0014] (a) CDR1 contains a variant of any amino acid sequence selected from SEQ ID NO:1, 4, 8 and 11, or any amino acid sequence selected from SEQ ID NO:1, 4, 8 and 11 with no more than two amino acid variations;

[0015] (b) CDR2 comprises a variant of any amino acid sequence selected from SEQ ID NO:2, 5, 9, 12, 14, or any amino acid sequence selected from SEQ ID NO:2, 5, 9, 12, 14, with no more than two amino acid variations; and

[0016] (c) CDR3 comprises a variant of any amino acid sequence selected from SEQ ID NO:3, 6, 7, 10, 13, or any amino acid sequence selected from SEQ ID NO:3, 6, 7, 10, 13 with no more than two amino acid variations.

[0017] The amino acid changes mentioned therein are the addition, deletion or substitution of amino acids. Preferably, the sdAb portion is a camel VHH, a partially humanized or fully humanized VHH, or a chimeric VHH.

[0018] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises

[0019] (i) CDR1 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, CDR2 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and CDR3 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:3 or SEQ ID NO:3.

[0020] (ii) CDR1 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:4 or SEQ ID NO:4, CDR2 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:5 or SEQ ID NO:5, and CDR3 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:6 or SEQ ID NO:6.

[0021] (iii) CDR1 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:4 or SEQ ID NO:4, CDR2 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:5 or SEQ ID NO:5, and CDR3 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:7 or SEQ ID NO:7.

[0022] (iv) CDR1 containing no more than 2 amino acid variations of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, CDR2 containing no more than 2 amino acid variations of the amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and CDR3 containing no more than 2 amino acid variations of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10.

[0023] (v) CDR1 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:11 or SEQ ID NO:11, CDR2 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:12 or SEQ ID NO:12, and CDR3 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:13 or SEQ ID NO:13; or (vi) CDR1 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, CDR2 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:14 or SEQ ID NO:14, and CDR3 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:10 or SEQ ID NO:10.

[0024] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing an amino acid sequence such as ATIFDRSVMX1, CDR2 containing an amino acid sequence such as RITSGGNX2X3, and CDR3 containing an amino acid sequence such as RGSVLLSX4YDY, wherein X1 is A or S; X2 is I or T; X3 is Y or N; and X4 is R or Q.

[0025] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence as shown in SEQ ID NO:4, CDR2 containing the amino acid sequence as shown in SEQ ID NO:5, and CDR3 containing the amino acid sequence as shown in SEQ ID NO:6.

[0026] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence as shown in SEQ ID NO:4, CDR2 containing the amino acid sequence as shown in SEQ ID NO:5, and CDR3 containing the amino acid sequence as shown in SEQ ID NO:7.

[0027] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:15, CDR2 containing the amino acid sequence shown in SEQ ID NO:16, and CDR3 containing the amino acid sequence shown in SEQ ID NO:7.

[0028] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:15, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:7.

[0029] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:15, CDR2 containing the amino acid sequence shown in SEQ ID NO:5, and CDR3 containing the amino acid sequence shown in SEQ ID NO:6.

[0030] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence as shown in SEQ ID NO:15, CDR2 containing the amino acid sequence as shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence as shown in SEQ ID NO:6.

[0031] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence as shown in SEQ ID NO:1, CDR2 containing the amino acid sequence as shown in SEQ ID NO:2, and CDR3 containing the amino acid sequence as shown in SEQ ID NO:3.

[0032] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:11, CDR2 containing the amino acid sequence shown in SEQ ID NO:12, and CDR3 containing the amino acid sequence shown in SEQ ID NO:13.

[0033] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises four framework regions arranged sequentially from the N-terminus to the C-terminus, namely FR1, FR2, FR3, and FR4. The four sequentially arranged framework regions comprise:

[0034] (i) FR1 containing the amino acid sequence shown in SEQ ID NO:36, FR2 containing the amino acid sequence shown in SEQ ID NO:37, FR3 containing the amino acid sequence shown in SEQ ID NO:38 and FR4 containing the amino acid sequence shown in SEQ ID NO:39;

[0035] (ii) FR1 containing the amino acid sequence as shown in SEQ ID NO:40, FR2 containing the amino acid sequence as shown in SEQ ID NO:41, FR3 containing the amino acid sequence as shown in SEQ ID NO:42 and FR4 containing the amino acid sequence as shown in SEQ ID NO:43;

[0036] (iii) FR1 containing the amino acid sequence as shown in SEQ ID NO:44, FR2 containing the amino acid sequence as shown in SEQ ID NO:41, FR3 containing the amino acid sequence as shown in SEQ ID NO:42 and FR4 containing the amino acid sequence as shown in SEQ ID NO:43;

[0037] (iv) FR1 containing the amino acid sequence as shown in SEQ ID NO:45, FR2 containing the amino acid sequence as shown in SEQ ID NO:41, FR3 containing the amino acid sequence as shown in SEQ ID NO:42 and FR4 containing the amino acid sequence as shown in SEQ ID NO:43;

[0038] (v) FR1 containing the amino acid sequence as shown in SEQ ID NO:46, FR2 containing the amino acid sequence as shown in SEQ ID NO:41, FR3 containing the amino acid sequence as shown in SEQ ID NO:42 and FR4 containing the amino acid sequence as shown in SEQ ID NO:43;

[0039] (vi) FR1 containing the amino acid sequence as shown in SEQ ID NO:47, FR2 containing the amino acid sequence as shown in SEQ ID NO:48, FR3 containing the amino acid sequence as shown in SEQ ID NO:49 and FR4 containing the amino acid sequence as shown in SEQ ID NO:50;

[0040] (vii) FR1 containing the amino acid sequence as shown in SEQ ID NO:47, FR2 containing the amino acid sequence as shown in SEQ ID NO:51, FR3 containing the amino acid sequence as shown in SEQ ID NO:52 and FR4 containing the amino acid sequence as shown in SEQ ID NO:50;

[0041] (viii) FR1 containing the amino acid sequence as shown in SEQ ID NO:53, FR2 containing the amino acid sequence as shown in SEQ ID NO:37, FR3 containing the amino acid sequence as shown in SEQ ID NO:38 and FR4 containing the amino acid sequence as shown in SEQ ID NO:60;

[0042] (ix) FR1 containing the amino acid sequence as shown in SEQ ID NO:54, FR2 containing the amino acid sequence as shown in SEQ ID NO:48, FR3 containing the amino acid sequence as shown in SEQ ID NO:49 and FR4 containing the amino acid sequence as shown in SEQ ID NO:50;

[0043] (x) contains FR1 with an amino acid sequence as shown in SEQ ID NO:55, FR2 with an amino acid sequence as shown in SEQ ID NO:41, FR3 with an amino acid sequence as shown in SEQ ID NO:42 and FR4 with an amino acid sequence as shown in SEQ ID NO:50;

[0044] (xi) contains FR1 with an amino acid sequence as shown in SEQ ID NO:56, FR2 with an amino acid sequence as shown in SEQ ID NO:57, FR3 with an amino acid sequence as shown in SEQ ID NO:58 and FR4 with an amino acid sequence as shown in SEQ ID NO:59.

[0045] (xii) FR1 containing the amino acid sequence as shown in SEQ ID NO:60, FR2 containing the amino acid sequence as shown in SEQ ID NO:61, FR3 containing the amino acid sequence as shown in SEQ ID NO:58 and FR4 containing the amino acid sequence as shown in SEQ ID NO:59;

[0046] (xiii) FR1 containing the amino acid sequence as shown in SEQ ID NO:60, FR2 containing the amino acid sequence as shown in SEQ ID NO:62, FR3 containing the amino acid sequence as shown in SEQ ID NO:63 and FR4 containing the amino acid sequence as shown in SEQ ID NO:59;

[0047] (xiv) FR1 containing the amino acid sequence as shown in SEQ ID NO:60, FR2 containing the amino acid sequence as shown in SEQ ID NO:57, FR3 containing the amino acid sequence as shown in SEQ ID NO:58, and FR4 containing the amino acid sequence as shown in SEQ ID NO:59; or

[0048] (xv) FR1 containing the amino acid sequence as shown in SEQ ID NO:60, FR2 containing the amino acid sequence as shown in SEQ ID NO:62, FR3 containing the amino acid sequence as shown in SEQ ID NO:64 and FR4 containing the amino acid sequence as shown in SEQ ID NO:59.

[0049] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises

[0050] (i) Any amino acid sequence in SEQ ID NO:18-27; or

[0051] (ii) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences in SEQ ID NO:18-27, for example, the amino acid sequences of SEQ ID NO:28, 29, 30, 31, 32, or 33.

[0052] In some embodiments, the CTLA-4 binding molecule of the present invention is also linked at the N-terminus or C-terminus of the sdAb portion to the Fc region of an immunoglobulin, for example to the Fc region of IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0053] In some embodiments, the CTLA-4 binding molecule of the present invention is a bispecific or multispecific antibody. Preferably, the bispecific antibody molecule binds to CTLA-4 and PD-1 or PD-L1, thereby blocking or inhibiting the binding of CTLA-4 to CD80 and / or CD86, and blocking or inhibiting the binding of PD-1 and PD-L1, thereby activating T cells.

[0054] In a second aspect, the present invention provides a method for preparing the CTLA-4 binding molecule of the present invention, the method comprising culturing a host cell containing a nucleic acid encoding the CTLA-4 binding molecule of the present invention or an expression vector containing said nucleic acid under conditions suitable for expressing a nucleic acid encoding the CTLA-4 binding molecule of the present invention, isolating said CTLA-4 binding molecule, and optionally further comprising recovering said CTLA-4 binding molecule from said host cell.

[0055] In a third aspect, the present invention provides an immunoconjugate comprising the CTLA-4 binding molecule of the present invention and other substances, such as cytotoxic agents.

[0056] In a fourth aspect, the present invention provides pharmaceutical compositions comprising the CTLA-4 binding molecule or immunoconjugate of the present invention, and optionally pharmaceutical excipients.

[0057] In some embodiments, the present invention provides a pharmaceutical composition comprising the CTLA-4 binding molecule or immunoconjugate of the present invention, as well as other therapeutic agents and optionally pharmaceutical excipients; preferably, the other therapeutic agents are selected from chemotherapeutic agents, other antibodies (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies), and cytotoxic agents.

[0058] In some embodiments, the present invention provides a combination product comprising the CTLA-4 binding molecule or immunoconjugate of the present invention, and one or more other therapeutic agents, such as chemotherapeutic agents, cytotoxic agents, other antibodies, preferably anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0059] In a fifth aspect, the present invention provides a method for preventing or treating tumors or infectious diseases in a subject or individual, comprising administering to the subject an effective amount of the CTLA-4 binding molecule, immune conjugate, pharmaceutical composition, or combination product of the present invention.

[0060] In some embodiments, the CTLA-4 binding molecule, immune conjugate, pharmaceutical composition, or combination product of the present invention prevents or treats tumors that are cancers, such as cancers with elevated CTLA-4 expression levels; or the CTLA-4 binding molecule, immune conjugate, pharmaceutical composition, or combination product of the present invention prevents or treats infectious diseases such as bacterial infections, viral infections, fungal infections, or protozoan infections, preferably said infectious diseases are chronic infections in which elevated CTLA-4 expression levels lead to immunodeficiency in the subject or individual.

[0061] In a sixth aspect, the present invention provides a kit for detecting CTLA-4 in a sample, the kit comprising the CTLA-4 binding molecule of the present invention for performing the following steps:

[0062] (a) Contacting the sample with the CTLA-4 binding molecule of the present invention; and

[0063] (b) Detecting the formation of the complex between the CTLA-4 binding molecule and CTLA-4; optionally, the CTLA-4 binding molecule is detectably labeled.

[0064] Therefore, it is possible to determine whether there is an elevated level of CTLA-4 expression in samples from subjects or individuals. Attached Figure Description

[0065] The preferred embodiments of the invention described in the following detailed description will be better understood when read in conjunction with the accompanying drawings. The drawings show presently preferred embodiments for illustrative purposes. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0066] Figure 1A The figure shows the results of detecting the specific binding activity of multiple anti-CTLA-4 VHH antibodies with CTLA-4 by FACS method after dilution at 1:1, 1:3, and 1:9. The negative control in the figure is the isotype control (IsotypeCtrl), which is a purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502). IPI is an abbreviation for the anti-CTLA-4 antibody ipilimumab; SS320 represents the blank control of Escherichia coli SS320; MFI represents the mean fluorescence intensity.

[0067] Figure 1B and Figure 1C The figure shows the results of detecting the specific binding activity of multiple anti-CTLA-4 VHH antibodies with CTLA-4 by FACS method after dilution at 1:1, 1:6, 1:36, and 1:216, respectively. The isotype control (IsotypeCtrl) is a purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502), IPI is an abbreviation for the anti-CTLA-4 antibody ipilimumab, cells only represent the blank control of Escherichia coli SS320, and MFI represents the mean fluorescence intensity.

[0068] Figure 2A and Figure 2B The figure shows the binding affinity of eight VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc to CTLA-4 on human CTLA-4CHO cells. The isotype control (IsotypeCtrl) in the figure refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); cells only represent the blank control of human CTLA-4CHO cells. Each VHH-Fc chimeric antibody is indicated by its respective VHH clone number.

[0069] Figure 3A and Figure 3B The FACS experiment demonstrated the blocking activity of the VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc against the binding of CTLA-4 and CD80. In the figure, the isotype control (Isotype Ctrl) refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); the cells only represent the blank control of human CTLA-4CHO cells. Each VHH-Fc chimeric antibody in the figure is indicated only by its respective VHH clone number.

[0070] Figure 4A and Figure 4B The FACS experiment demonstrated the blocking activity of the VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc against the binding of CTLA-4 and CD86. In the figure, the isotype control (Isotype Ctrl) refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); the cells only represent the blank control of human CTLA-4CHO cells. Each VHH-Fc chimeric antibody in the figure is indicated only by its respective VHH clone number.

[0071] Figure 5A and Figure 5B The figure shows the binding affinity of eight VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc to monkey CTLA-4CHO cells. The isotype control (IsotypeCtrl) in the figure refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); the cells shown represent blank controls of monkey CTLA-4CHO cells. Each VHH-Fc chimeric antibody is indicated by its respective VHH clone number.

[0072] Figure 6A The figure shows the effect of five VHH-Fc chimeric antibodies obtained by fusing the five VHH molecules screened in Example 3 with Fc on the IL-2 secretion level of PBMC cells after activation. In the figure, the isotype control (Isotype Ctrl) refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); blank controls consisting only of PBMC cells are shown. Each VHH-Fc chimeric antibody is represented by its respective VHH clone number in the figure.

[0073] Figure 6B The figure shows the effect of three VHH-Fc chimeric antibodies obtained by fusing three additional VHH molecules screened in Example 3 with Fc on the IL-2 secretion level of PBMC cells after activation. In the figure, the isotype control (Isotype Ctrl) refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); blank controls consisting only of PBMC cells are shown. Each VHH-Fc chimeric antibody is represented by its respective VHH clone number in the figure.

[0074] Figure 7AThe figure shows the effect of five VHH-Fc chimeric antibodies obtained by fusing the five VHH molecules screened in Example 3 with Fc on the secretion of TNFα in activated PBMC cells. In the figure, the isotype control (Isotype Ctrl) refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); blank controls consisting only of PBMC cells are shown. Each VHH-Fc chimeric antibody is represented by its respective VHH clone number.

[0075] Figure 7B The figure shows the effect of three VHH-Fc chimeric antibodies obtained by fusing three additional VHH molecules screened in Example 3 with Fc on the secretion of TNFα in activated PBMC cells. In the figure, the isotype control (Isotype Ctrl) refers to the purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502); the blank control is represented by PBMC cells only. Each VHH-Fc chimeric antibody is represented by its respective VHH clone number in the figure.

[0076] Figure 8 The binding affinity of the humanized VHH-Fc chimeric antibody to human CTLA-4 was demonstrated by FACS experiments. In the figure, the NB25B-17-Fc chimeric antibody and the NB25gb-1-Fc chimeric antibody are represented by their respective VHH clone numbers, namely NB25B-17 and NB25gb-1.

[0077] Figure 9 The figure shows the blocking activity of the humanized VHH-Fc chimeric antibody against CTLA-4 and CD80 binding, as identified by FACS. The NB25B-17-Fc chimeric antibody and the NB25gb-1-Fc chimeric antibody are represented by their respective VHH clone numbers, namely NB25B-17 and NB25gb-1, respectively.

[0078] Figure 10 The figure shows the blocking activity of the humanized VHH-Fc chimeric antibody against the binding of CTLA-4 and CD86, as identified by FACS. The NB25B-17-Fc chimeric antibody and the NB25gb-1-Fc chimeric antibody are represented by their respective VHH clone numbers, namely NB25B-17 and NB25gb-1, respectively.

[0079] Figure 11The binding affinity of the humanized VHH-Fc chimeric antibody to monkey CTLA-4 was demonstrated by FACS experiments. In the figure, the NB25B-17-Fc chimeric antibody and the NB25gb-1-Fc chimeric antibody are represented by their respective VHH clone numbers, namely NB25B-17 and NB25gb-1. Detailed Implementation

[0080] Unless otherwise specified, 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 pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0081] I. Definition

[0082] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0083] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0084] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0085] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the variable region consists of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0086] The term "antibody" is used in the broadest sense herein to refer to a protein containing an antigen-binding site, encompassing a wide range of natural and artificial antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments. Preferably, the antibodies of the present invention are single-domain antibodies or heavy-chain antibodies.

[0087] An "antibody fragment" refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific or multispecific antibodies formed from antibody fragments.

[0088] As used herein, the term “epitope” refers to the portion of an antigen (e.g., human CTLA-4) that specifically interacts with an antibody molecule.

[0089] An antibody exhibiting the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for measuring binding affinity and / or specificity. In one embodiment, the reference antibody is ipilimumab.

[0090] The complementarity-determining region (CDR) or CDR is a region within the antibody's variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. Heavy chain CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, 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 antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (http: / / imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0091] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0092] The CDR can also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any of the CDRs in the examples of this invention). In one embodiment, the CDR of the single-domain antibody of this invention is determined according to the AbM numbering scheme.

[0093] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR (including heavy chain variable region residues), it means the numbering position according to the AbM numbering system.

[0094] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, or AbM may be substituted with conserved amino acid residues.

[0095] The term "single-domain antibody" generally refers to an antibody in which a single variable domain (e.g., a heavy chain variable domain (VH) or light chain variable domain (VL), a heavy chain variable domain derived from camelid heavy chain antibodies, or a VH-like single domain (v-NAR) derived from fish IgNARs) is sufficient to confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks) (WO 2005 / 035572). Camelid single-domain antibodies, also referred to herein as "camelid VHH," consist of only one heavy chain variable region, comprising a single chain from C-terminus to N-terminus: FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1, and can be naturally produced by camels or generated through genetic engineering. Single-domain antibodies are the smallest known units that can bind to a target antigen.

[0096] As used herein, the term "heavy-chain antibody (hcAb)" refers to an antibody that does not have a light chain and may contain VH-CH2-CH3 or VH-CH1-CH2-CH3 from the N-terminus to the C-terminus; it may form a homodimer, such as a heavy-chain dimer antibody that does not have a light chain. The heavy-chain antibody of the present invention may contain VH from a standard antibody or VH from a single-domain antibody. For example, the VH in the heavy-chain antibody of the present invention may be a single-domain antibody.

[0097] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigenic epitopes. In one embodiment, this document provides such a bispecific antibody having binding specificity against a first antigen and a second antigen.

[0098] The term "effective function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0099] The term "chimeric antibody" refers to an antibody molecule in which (a) a constant region or a portion thereof is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered class, effector function, and / or species of constant region, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), which imparts new properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged with a variable region having different or altered antigen specificity. For example, a camel antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulins. By replacing it with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced antigenicity in humans, as compared to the original camel antibody.

[0100] "Humanized" antibodies are chimeric antibodies comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in a humanized antibody correspond to those in a nonhuman antibody, and all or substantially all of the FRs correspond to those in a human antibody. Humanized antibodies may optionally contain at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized.

[0101] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, utilizing a human antibody library or other human antibody encoding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0102] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes 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 carbonyl terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as 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.

[0103] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6) th (ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to provide antigen binding specificity.

[0104] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biomembrane interferometry, or other conventional binding assays known in the art.

[0105] The term "immune checkpoint molecules" refers to a class of inhibitory signaling molecules present in the immune system. These molecules regulate the persistence and intensity of immune responses in peripheral tissues to prevent tissue damage and participate in maintaining tolerance to self-antigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4):252-264). Studies have found that one reason why tumor cells can evade the body's immune system and proliferate uncontrollably is by utilizing the inhibitory signaling pathways of immune checkpoint molecules, thereby inhibiting T lymphocyte activity and preventing T lymphocytes from effectively exerting their killing effect on tumors (Yao S, Zhu Y and Chen L., Advances intargeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2):130-146). Immune checkpoint molecules include, but are not limited to, CTLA-4, programmed cell death 1 (PD-1), PD-L2, LAG-3, and TIM-3.

[0106] The term "co-stimulatory molecule" refers to a corresponding binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell (e.g., but not limited to proliferation). Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28. In some embodiments, the "co-stimulatory molecule" is CD28, OX40, GITR, 4-1BB (i.e., CD137), and / or CD27.

[0107] The term "cytokine" is a general term for proteins released by one group of cells that act as intercellular mediators to another cell. Examples of such cytokines include lymphokines, monokines, interleukins (ILs) 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, and IL-15; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors, including gamma interferon.

[0108] "Immune conjugates" are antibodies conjugated to one or more other substances (including but not limited to cytotoxic agents or markers).

[0109] The terms "inhibition" or "blocking" refer to reducing certain parameters (e.g., activity) of a given molecule (e.g., an immune checkpoint molecule). For example, this term includes substances that inhibit the activity of a given molecule (e.g., CTLA-4) by at least 5%, 10%, 20%, 30%, 40%, or more. Therefore, the inhibition need not be 100%.

[0110] The “functional Fc region” possesses the “effective functions” of the native Fc region. Exemplary “effective functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require the Fc region to be coupled with a binding domain (e.g., antibody variable domain) and can be assessed using various assays, such as those disclosed herein.

[0111] "Effective functions" refer to biological activities that can be attributed to the Fc region of an antibody and vary with antibody isotypes. 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 receptors); and B cell activation.

[0112] The term "effective amount" refers to such an amount or dose of the CTLA-4 binding molecule, conjugate, or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; weight, age, and general health condition; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0113] "Therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective amount of an antibody or antibody fragment, or its conjugate or composition, can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or antibody fraction to elicit the desired response in the individual. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment, or its conjugate or composition, are less than the therapeutically beneficial effects. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits measurable parameters (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0114] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to subjects before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0115] The terms “individual” or “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human being.

[0116] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and liquid tumors.

[0117] The terms “cancer” and “cancerous” refer to physiological disorders in mammals where cell growth is not regulated.

[0118] The term "tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when used in this article.

[0119] "Tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when this evasion weakens, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0120] The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

[0121] The term "chronic infection" refers to an infection in which an infectious agent (e.g., pathogens such as viruses, bacteria, protozoa such as parasites, fungi, or the like) has induced an immune response in the infected host but has not yet been cleared or eliminated from the host as in the case of an acute infection. Chronic infections can be persistent, latent, or slow-growing. In one embodiment, the CTLA-4 binding molecule of the present invention is used to treat a chronic infectious disease caused by Mycobacterium tuberculosis (MTB)—tuberculosis.

[0122] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of probes or antibodies by reacting with another directly labeled reagent. Examples of indirect labeling include the detection of a first antibody using a fluorescently labeled second antibody and the end labeling of a biotinylated DNA probe, making it detectable with a fluorescently labeled streptomycin protein.

[0123] "Separated" CTLA-4 binding molecules refer to those that have been separated from components of their native environment. In some embodiments, CTLA-4 binding molecules are purified to a purity exceeding 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0124] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include those contained in cells that normally contain the nucleic acid molecule, but which are located extrachromosomally or at a chromosomal location different from their natural chromosomal location. "Isolated nucleic acid encoding a CTLA-4 binding molecule" refers to one or more nucleic acid molecules, the strand or fragment thereof encoding a CTLA-4 binding molecule, including such nucleic acid molecules in a single or separate vector, and such nucleic acid molecules present at one or more locations within a host cell.

[0125] The following is a calculation of sequence identity between sequences.

[0126] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in 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 purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0127] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0128] Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0129] Additionally or alternatively, the nucleic acid and protein sequences described herein may be further used as “query sequences” to perform searches against public databases, for example, to identify other family member sequences or related sequences.

[0130] As used herein, the term "hybridization under low, medium, high, or very high strictness conditions" describes the hybridization and washing conditions. Instructions for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which are incorporated herein by reference. Aqueous and non-aqueous methods are described in this reference, and either method may be used. In some embodiments, the specific hybridization conditions mentioned herein are as follows: 1) Low-toughness hybridization conditions are performed at approximately 45°C in 6X sodium chloride / sodium citrate (SSC), followed by two washes at at least 50°C (for low-toughness conditions, the washing temperature can be increased to 55°C) in 0.2X SSC, 0.1% SDS; 2) Medium-toughness hybridization conditions are performed at approximately 45°C in 6X SSC, followed by one or more washes at 60°C in 0.2X SSC, 0.1% SDS; 3) High-toughness hybridization conditions are performed at approximately 45°C in 6X SSC, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS; and preferably 4) Very high-toughness hybridization conditions are performed at 65°C in 0.5M sodium phosphate, 7% SDS, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS. Very high-toughness condition (4) is the preferred condition and, unless otherwise stated, should be used.

[0131] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0132] The term "pharmaceutical excipients" refers to diluents, adjuvants (such as Freund's adjuvants (complete and incomplete)), carriers, excipients, or stabilizers that are applied together with the active substance.

[0133] When used herein, "treatment" means to slow, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay disease development or to slow disease progression.

[0134] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0135] The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors (e.g., cancer) and infections (e.g., chronic infections), including chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

[0136] "Chemotherapy agents" include chemical compounds useful in the treatment of cancer, including but not limited to antitumor agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; antiestrogens; antiandrogens; and nonsteroidal antiandrogens, etc. Examples of chemotherapeutic agents are those disclosed in WO2015 / 153513 or WO2016 / 028672 or WO2015 / 138920.

[0137] As used in this article, the term "immunomodulator" refers to natural or synthetic active agents or drugs that inhibit or modulate immune responses. Immune responses can be humoral or cellular. Immunomodulators include inhibitors of immune checkpoint molecules and activators of co-stimulatory molecules.

[0138] The term "small molecule drug" refers to low molecular weight organic compounds that can regulate biological processes.

[0139] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Examples of cytotoxic agents can be found in those disclosed in WO2015 / 153513, WO2016 / 028672 or WO2015 / 138920.

[0140] The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, such as parasites, at the applied concentration and dosing interval. When used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoan agents. In one specific aspect, the anti-infective active agent is non-toxic to the host at the applied concentration and dosing interval.

[0141] Antibacterial agents, or antibacterial actives, can be broadly classified as bactericidal (i.e., directly killing bacteria) or bacteriostatic (i.e., inhibiting cell division). Antibacterial agents can be further subdivided into narrow-spectrum antibacterial agents (i.e., affecting only a small number of bacterial subtypes, such as Gram-negative bacteria) or broad-spectrum antibacterial agents (i.e., affecting a wide range of species). Examples include amikacin, gentamicin, geldmycin, atrazine, mupirocin, nitrofurantoin, pyrazinamide, quinupristin / dalfopristin, rifampin / isofopristin, or tinidazole.

[0142] The term "antiviral agent" includes any substance that inhibits or eliminates viral growth, pathogenicity, and / or survival. This includes, for example, acyclovir, cidofovir, zidovudine, doxorinosine (ddI, VIDEX), zalcitabine (ddC, HIVID), stavudine (d4T, ZERIT), lamivudine (3TC, EPIVIR), abacavir (ZIAGEN), emtricitabine (EMTRIVA), etc.

[0143] The term "antifungal agent" includes any substance that inhibits or eliminates the growth, pathogenicity, and / or survival of fungi. This includes, for example, natamycin, sclerotinib, ferulic acid, nystatin, amphotericin B, candisin, patchouli, neem seed oil, coconut oil, etc.

[0144] The term "antiprotozoan agent" includes any substance that inhibits or eliminates the growth, disease, and / or survival of protozoan organisms (such as parasites). Examples of antiprotozoan agents include antimalarial agents such as quinine and quinidine.

[0145] Exemplary antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents are listed for example, WO2010 / 077634. ​​Anti-infective agents are also listed for example, WO2014 / 008218, WO2016 / 028672, or WO2015 / 138920.

[0146] The term "combination product" refers to a fixed or non-fixed combination or a kit for combination administration in the form of a dose unit, wherein two or more therapeutic agents can be administered independently at the same time or separately at time intervals, particularly where these time intervals allow the combined therapeutic agents to exhibit synergy, e.g., a co-existing effect. The term "fixed combination" refers to the simultaneous administration of the CTLA-4 binding molecule and combination partner (e.g., other therapeutic agents, such as anti-PD-1 antibodies or anti-PD-L1 antibodies) of the present invention to a patient in the form of a single entity or dose. The term "non-fixed combination" means that the CTLA-4 binding molecule and combination partner (e.g., other therapeutic agents, such as anti-PD-1 antibodies or anti-PD-L1 antibodies) of the present invention are administered to a patient as separate entities simultaneously, in parallel, or sequentially, without a specific time limit, wherein such administration provides a therapeutically effective level of both therapeutic agents in the patient. The latter also applies to cocktail therapies, such as the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

[0147] The terms "combination therapy" or "combination therapy" refer to the administration of two or more therapeutic agents to treat cancer or infection as described in this disclosure. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed proportion of active ingredients. Alternatively, such administration includes co-administration, separate administration, or sequential administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. In some embodiments, administration also includes using each type of therapeutic agent at substantially the same time, or at different times in a sequential manner. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0148] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of the nucleic acid to which they are effectively linked. Such vectors are referred to herein as "expression vectors."

[0149] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. This includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells. Host cells are any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0150] "Subject / Patient Sample" refers to a collection of cells, tissues, or body fluids obtained from a patient or subject. The source of tissue or cell samples can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds naturally occurring and not contaminated with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples in this document include, but are not limited to, tumor biopsies, fine-needle aspirations, bronchoalveolar lavage fluid, 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 exhibiting tumor-like characteristics, and preserved tumor samples, such as formalin-fixed, paraffin-embedded, or frozen tumor samples.

[0151] The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such a therapeutic product.

[0152] II. The CTLA-4 binding molecule of the present invention

[0153] The CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) moiety that specifically binds to CTLA-4, wherein the sdAb moiety comprises three complementarity-determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2, and CDR3, wherein:

[0154] (a) CDR1 contains a variant of any amino acid sequence selected from SEQ ID NO:1, 4, 8 and 11, or any amino acid sequence selected from SEQ ID NO:1, 4, 8 and 11 with no more than two amino acid variations;

[0155] (b) CDR2 comprises a variant of any amino acid sequence selected from SEQ ID NO:2, 5, 9, 12, 14, or any amino acid sequence selected from SEQ ID NO:2, 5, 9, 12, 14, with no more than two amino acid variations; and

[0156] (c) CDR3 comprises a variant of any amino acid sequence selected from SEQ ID NO:3, 6, 7, 10, 13, or any amino acid sequence selected from SEQ ID NO:3, 6, 7, 10, 13 with no more than two amino acid variations.

[0157] The amino acid change mentioned therein is the addition, deletion, or substitution of an amino acid; for example, the amino acid change is a conserved amino acid substitution.

[0158] In some embodiments, the CTLA-4 binding molecule of the present invention binds to mammalian CTLA-4, such as human CTLA-4 or monkey CTLA-4. For example, the CTLA-4 binding molecule of the present invention specifically binds to epitopes (e.g., linear or conformational epitopes) on CTLA-4. In some embodiments, the CTLA-4 binding molecule binds to one or more extracellular domains of CTLA-4.

[0159] In some embodiments, the CTLA-4 binding molecule of the present invention has one or more of the following properties:

[0160] (1) Binding CTLA-4 with high affinity, such as human CTLA-4, for example, the Kd of the binding between the CTLA-4 binding molecule and CTLA-4 is about 10. -5 M to approximately 10 -12 M, preferably, about 10 -7 M to approximately 10 -12 M;

[0161] (2) Block the related activities of CTLA-4, for example, block the binding of CTLA-4 to CD80 and / or CD86;

[0162] (3) Induces the secretion of IL-2 and / or TNF-α;

[0163] (4) Keep T cells in an activated state.

[0164] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) portion that specifically binds to CTLA-4, said sdAb portion comprising CDR1 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:1 or SEQ ID NO:1, CDR2 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:2 or SEQ ID NO:2, and CDR3 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:3 or SEQ ID NO:3, wherein said amino acid variations are additions, deletions, or substitutions of amino acids, for example, said amino acid variations are conserved amino acid substitutions.

[0165] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) portion that specifically binds to CTLA-4, said sdAb portion comprising CDR1 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:4 or SEQ ID NO:4, CDR2 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:5 or SEQ ID NO:5, and CDR3 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:6 or SEQ ID NO:6, wherein said amino acid variations are additions, deletions, or substitutions of amino acids, for example, said amino acid variations are conserved amino acid substitutions.

[0166] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) portion that specifically binds to CTLA-4, said sdAb portion comprising CDR1 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:4 or SEQ ID NO:4, CDR2 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:5 or SEQ ID NO:5, and CDR3 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:7 or SEQ ID NO:7, wherein said amino acid variations are additions, deletions, or substitutions of amino acids, for example, said amino acid variations are conserved amino acid substitutions.

[0167] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) portion that specifically binds to CTLA-4, said sdAb portion comprising CDR1 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, CDR2 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:9 or SEQ ID NO:9, and CDR3 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:10 or SEQ ID NO:10, wherein said amino acid variations are additions, deletions, or substitutions of amino acids, for example, said amino acid variations are conserved amino acid substitutions.

[0168] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) portion that specifically binds to CTLA-4, said sdAb portion comprising CDR1 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:11 or SEQ ID NO:11, CDR2 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:12 or SEQ ID NO:12, and CDR3 containing no more than two amino acid variations of the amino acid sequence SEQ ID NO:13 or SEQ ID NO:13, wherein said amino acid variations are additions, deletions, or substitutions of amino acids, for example, said amino acid variations are conserved amino acid substitutions.

[0169] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one single-domain antibody (sdAb) portion that specifically binds to CTLA-4, said sdAb portion comprising CDR1 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:8 or SEQ ID NO:8, CDR2 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:14 or SEQ ID NO:14, and CDR3 containing no more than two amino acid variations of amino acid sequence SEQ ID NO:10 or SEQ ID NO:10, wherein said amino acid variations are additions, deletions, or substitutions of amino acids, for example, said amino acid variations are conserved amino acid substitutions.

[0170] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence ATIFDRSVMX1, CDR2 containing the amino acid sequence RITSGGNX2X3, and CDR3 containing the amino acid sequence RGSVLLSX4YDY, wherein X1 is A or S; X2 is I or T; X3 is Y or N; and X4 is R or Q.

[0171] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:4, CDR2 containing the amino acid sequence SEQ ID NO:5, and CDR3 containing the amino acid sequence SEQ ID NO:6.

[0172] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:4, CDR2 containing the amino acid sequence SEQ ID NO:5, and CDR3 containing the amino acid sequence SEQ ID NO:7.

[0173] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:15, CDR2 containing the amino acid sequence SEQ ID NO:16, and CDR3 containing the amino acid sequence SEQ ID NO:7.

[0174] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:15, CDR2 containing the amino acid sequence SEQ ID NO:17, and CDR3 containing the amino acid sequence SEQ ID NO:7.

[0175] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:15, CDR2 containing the amino acid sequence SEQ ID NO:5, and CDR3 containing the amino acid sequence SEQ ID NO:6.

[0176] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:15, CDR2 containing the amino acid sequence SEQ ID NO:17, and CDR3 containing the amino acid sequence SEQ ID NO:6.

[0177] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: CDR1 containing the amino acid sequence SEQ ID NO:1, CDR2 containing the amino acid sequence SEQ ID NO:2, and CDR3 containing the amino acid sequence SEQ ID NO:3.

[0178] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: wherein the sdAb portion comprises CDR1 containing the amino acid sequence SEQ ID NO:11, CDR2 containing the amino acid sequence SEQ ID NO:12, and CDR3 containing the amino acid sequence SEQ ID NO:13.

[0179] In some embodiments, the sdAb portion of the CTLA-4 binding molecule of the present invention comprises: the sdAb portion includes four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, wherein:

[0180] (i) FR1 containing the amino acid sequence SEQ ID NO:36, FR2 containing the amino acid sequence SEQ ID NO:37, FR3 containing the amino acid sequence SEQ ID NO:38 and FR4 containing the amino acid sequence SEQ ID NO:39;

[0181] (ii) FR1 containing the amino acid sequence SEQ ID NO:40, FR2 containing the amino acid sequence SEQ ID NO:41, FR3 containing the amino acid sequence SEQ ID NO:42 and FR4 containing the amino acid sequence SEQ ID NO:43;

[0182] (iii) FR1 containing the amino acid sequence SEQ ID NO:44, FR2 containing the amino acid sequence SEQ ID NO:41, FR3 containing the amino acid sequence SEQ ID NO:42 and FR4 containing the amino acid sequence SEQ ID NO:43;

[0183] (iv) FR1 containing the amino acid sequence SEQ ID NO:45, FR2 containing the amino acid sequence SEQ ID NO:41, FR3 containing the amino acid sequence SEQ ID NO:42 and FR4 containing the amino acid sequence SEQ ID NO:43;

[0184] (v) FR1 containing the amino acid sequence SEQ ID NO:46, FR2 containing the amino acid sequence SEQ ID NO:41, FR3 containing the amino acid sequence SEQ ID NO:42 and FR4 containing the amino acid sequence SEQ ID NO:43;

[0185] (vi) FR1 containing the amino acid sequence SEQ ID NO:47, FR2 containing the amino acid sequence SEQ ID NO:48, FR3 containing the amino acid sequence SEQ ID NO:49 and FR4 containing the amino acid sequence SEQ ID NO:50;

[0186] (vii) FR1 containing the amino acid sequence SEQ ID NO:47, FR2 containing the amino acid sequence SEQ ID NO:51, FR3 containing the amino acid sequence SEQ ID NO:52 and FR4 containing the amino acid sequence SEQ ID NO:50;

[0187] (viii) FR1 containing the amino acid sequence SEQ ID NO:53, FR2 containing the amino acid sequence SEQ ID NO:37, FR3 containing the amino acid sequence SEQ ID NO:38 and FR4 containing the amino acid sequence SEQ ID NO:60;

[0188] (ix) FR1 containing the amino acid sequence SEQ ID NO:54, FR2 containing the amino acid sequence SEQ ID NO:48, FR3 containing the amino acid sequence SEQ ID NO:49 and FR4 containing the amino acid sequence SEQ ID NO:50;

[0189] (x) FR1 containing the amino acid sequence SEQ ID NO:55, FR2 containing the amino acid sequence SEQ ID NO:41, FR3 containing the amino acid sequence SEQ ID NO:42 and FR4 containing the amino acid sequence SEQ ID NO:50;

[0190] (xi) FR1 containing the amino acid sequence SEQ ID NO:56, FR2 containing the amino acid sequence SEQ ID NO:57, FR3 containing the amino acid sequence SEQ ID NO:58, and FR4 containing the amino acid sequence SEQ ID NO:59; (xii) FR1 containing the amino acid sequence SEQ ID NO:60, FR2 containing the amino acid sequence SEQ ID NO:61, FR3 containing the amino acid sequence SEQ ID NO:58, and FR4 containing the amino acid sequence SEQ ID NO:59;

[0191] (xiii) FR1 containing the amino acid sequence SEQ ID NO:60, FR2 containing the amino acid sequence SEQ ID NO:62, FR3 containing the amino acid sequence SEQ ID NO:63 and FR4 containing the amino acid sequence SEQ ID NO:59;

[0192] (xiv) FR1 containing the amino acid sequence SEQ ID NO:60, FR2 containing the amino acid sequence SEQ ID NO:57, FR3 containing the amino acid sequence SEQ ID NO:58, and FR4 containing the amino acid sequence SEQ ID NO:59; or

[0193] (xv) FR1 containing the amino acid sequence SEQ ID NO:60, FR2 containing the amino acid sequence SEQ ID NO:62, FR3 containing the amino acid sequence SEQ ID NO:64 and FR4 containing the amino acid sequence SEQ ID NO:59.

[0194] In some embodiments, the CTLA-4 binding molecule of the present invention includes at least one CTLA-4-specific single-domain antibody (sdAb) moiety, said sdAb moiety being a camel VHH. In some embodiments, said camel VHH comprises or is composed of the following sequences:

[0195] (i) Any amino acid sequence in SEQ ID NO:18-27;

[0196] (ii) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences in SEQ ID NO: 18-27, for example, the amino acid sequences of SEQ ID NO: 28, 29, 30, 31, 32, or 33; or

[0197] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid variations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any of SEQ ID NO:18-27, preferably, the amino acid variations do not occur in the CDR region.

[0198] In some embodiments, the CTLA-4 binding molecule of the present invention comprises at least one CTLA-4-specific single-domain antibody (sdAb) moiety, said sdAb moiety being a partially or fully humanized VHH, or a chimeric VHH. Different methods for humanizing camel VHHs are known to those skilled in the art, such as those reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13:1619-1633).

[0199] In some implementations, the humanized VHH

[0200] (i) Containing or consisting of an amino acid sequence of SEQ ID NO:28, 29, 30, 31, 32 or 33;

[0201] (ii) Containing or consisting of 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 of SEQ ID NO: 28, 29, 30, 31, 32, or 33; or

[0202] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid variations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 28, 29, 30, 31, 32, or 33, preferably, the amino acid variations do not occur in the CDR region.

[0203] In some embodiments, the CTLA-4 binding molecule of the present invention is linked at the N-terminus or C-terminus of its sdAb portion to the Fc region of an immunoglobulin, optionally via an amino acid linker, for example, via an amino acid linker of length between 1 and 20 amino acids. In some embodiments, the amino acid linker is at least 90% glycine and / or serine amino acids. In some embodiments, the Fc region is derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1. In some embodiments, the Fc region is derived from human IgG1.

[0204] In some embodiments of the invention, the amino acid changes described herein include amino acid substitution, insertion, or deletion. Preferably, the amino acid changes described herein are amino acid substitutions, and more preferably, conservative substitutions.

[0205] In a preferred embodiment, the amino acid change described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid change described in this invention occurs in a region outside the VHH. In some embodiments, the substitution is a conservative substitution. A conservative substitution means that an amino acid is substituted by another amino acid within the same class, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid. Exemplary substitutions are shown in Table 1 below:

[0206] Table 1

[0207] Original residues Exemplary replacement Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu, Val; Met; Ala; Phe; Leucine Leu Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0208] In some embodiments, the CTLA-4 binding molecules provided herein are modified to increase or decrease their degree of glycosylation. The addition or deletion of glycosylation sites in the CTLA-4 binding molecule can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the CTLA-4 binding molecule contains an Fc region, the sugars linked to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, for example, removing the fucose module to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the CTLA-4 binding molecule provided herein to create Fc region variants to enhance the efficacy of, for example, the CTLA-4 binding molecules of the present invention, in treating cancer or infectious diseases.

[0209] In some embodiments, the CTLA-4 binding molecule of the present invention is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity against CTLA-4 and a second binding specificity against PD-1 or PD-L1. In one embodiment, the bispecific antibody molecule binds to both CTLA-4 and PD-1. In another embodiment, the bispecific antibody molecule binds to both CTLA-4 and PD-L1. The multispecific antibody molecule may, for example, be a trispecific antibody molecule comprising a first binding specificity against CTLA-4 and second and third binding specificities against one or more of the following molecules: PD-1, PD-L1, 41BB, OX40, or LAG-3.

[0210] III. Immunoconjugates

[0211] This invention also relates to CTLA-4 binding molecules (“immunoconjugates”) conjugated with other substances. In some embodiments, the other substances are, for example, therapeutic agents (such as cytotoxic agents). Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (such as chemotherapeutic agents) suitable for forming immunoconjugates are known in the art. For example, cytotoxic agents include, but are not limited to: radioactive isotopes; growth inhibitors; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known antitumor or anticancer agents.

[0212] Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immune conjugates also include, for example, WO2015 / 153513 or WO2015 / 138920.

[0213] The CTLA-4 binding molecule of the present invention can also be linked to a solid support, which is particularly useful for immunoassays or the purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0214] In some embodiments, the immune conjugate is used to prevent or treat tumors. In some embodiments, the tumor is cancer. In some embodiments, the immune conjugate is used to prevent or treat infections, such as chronic infections, including bacterial infections, viral infections, fungal infections, protozoan infections, etc.

[0215] IV. The nucleic acid of the present invention and the host cell containing it.

[0216] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned CTLA-4 binding molecules or fragments thereof or any strand thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. 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 preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0217] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:18-33, 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 an amino acid sequence selected from any one of SEQ ID NO:18-33.

[0218] The present invention also covers nucleic acids that hybridize under stringent conditions with or encode polypeptide sequences having one or more amino acid substitutions (e.g., conserved substitutions), deletions, or insertions compared to the following nucleic acids: nucleic acids comprising nucleic acid sequences encoding amino acid sequences selected from any one of SEQ ID NO:18-33; or nucleic acids comprising nucleic acid sequences encoding amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acid sequences selected from any one of SEQ ID NO:18-33.

[0219] In one embodiment, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In one embodiment, the vector is a pHEN1 vector.

[0220] Once the expression vector or DNA sequence for expression has been prepared, it can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells.

[0221] Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0222] In one embodiment, a host cell comprising the polynucleotides of the present invention is provided. In some embodiments, a host cell comprising 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 antibody preparation. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*. The host cell can also be a eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as a host. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), 293 cells, young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine 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, and myeloma cell lines such as Y0, NSO, P3X63, and Sp2 / 0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a 293 cell.

[0223] V. Production and purification of the CTLA-4 binding molecule of the present invention

[0224] In one embodiment, the present invention provides a method for preparing a CTLA-4 binding molecule, wherein the method includes culturing a host cell containing a nucleic acid encoding the CTLA-4 binding molecule or an expression vector containing the nucleic acid under conditions suitable for expressing a nucleic acid encoding the CTLA-4 binding molecule, and optionally isolating the CTLA-4 binding molecule. In one embodiment, the method further includes recovering the CTLA-4 binding molecule from the host cell (or host cell culture medium).

[0225] To recombinantly generate the CTLA-4 binding molecule of the present invention, the nucleic acid encoding the CTLA-4 binding molecule of the present invention is first isolated, and said nucleic acid is inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using routine procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the CTLA-4 binding molecule of the present invention.

[0226] The CTLA-4 binding molecule of the present invention, prepared as described herein, can be purified using known prior art techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the CTLA-4 binding molecule of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0227] VI. The method for determining the activity of CTLA-4 binding molecules of the present invention

[0228] The CTLA-4 binding molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the CTLA-4 binding molecules of the present invention can be tested, for example by known methods such as ELISA, Western blotting, etc. Binding to CTLA-4 can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, the binding of the CTLA-4 binding molecules of the present invention to CTLA-4 is determined using SPR or biofilm layer interferometry.

[0229] The present invention also provides a assay for identifying biologically active CTLA-4 binding molecules. Biological activity may include, for example, binding to cell surface CTLA-4 (e.g., human CTLA-4, monkey CTLA-4), inhibition of CTLA-4 / CD80 or CTLA-4 / CD86 binding, and promotion of IL-2 and / or TNF-α secretion.

[0230] Cells used for any of the above in vitro assays include naturally expressed CTLA-4 or modified CTLA-4 expressed cell lines. The modified CTLA-4 expressed cell line is a cell line that does not normally express CTLA-4 but expresses CTLA-4 after being transfected with DNA encoding CTLA-4.

[0231] It is understood that the immunoconjugates of the present invention can be used to replace the CTLA-4 binding molecule for any of the above-described assays.

[0232] VII. Pharmaceutical compositions and pharmaceutical preparations

[0233] In some embodiments, the present invention provides compositions comprising any CTLA-4 binding molecule or its immunoconjugate described herein, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises a combination of the CTLA-4 binding molecule or its immunoconjugate of the present invention and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators, preferably anti-PD-1 antibodies or anti-PD-L1 antibodies).

[0234] In some embodiments, the composition is used to prevent or treat tumors. In some embodiments, the tumor is cancer. In some embodiments, the composition is used to prevent or treat infections, such as chronic infections, including bacterial infections, viral infections, fungal infections, protozoan infections, etc.

[0235] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a CTLA-4 binding molecule or an immunoconjugate thereof and / or compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding a CTLA-4 binding molecule. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0236] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Pharmaceutical carriers suitable for this invention can be sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For information on the use and applications of excipients, see also "Handbook of Pharmaceutical Excipients," 5th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago. If desired, the compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, saccharin.

[0237] Pharmaceutical formulations containing the CTLA-4 binding molecule described herein can be prepared by mixing the CTLA-4 binding molecule of the present invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0238] The pharmaceutical compositions or formulations of the present invention may further comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anticancer or anti-infective active ingredients, such as chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, etc. The active ingredients are suitably combined in amounts effective for the intended use.

[0239] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the CTLA-4 binding molecule of the present invention, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0240] VIII. Combination products or reagent kits

[0241] In some embodiments, the present invention also provides a combination product comprising the CTLA-4 binding molecule of the present invention or its antigen-binding fragment, or its immunoconjugate, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective agents, small molecule drugs, or immunomodulators). In some embodiments, other antibodies include, for example, anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0242] In some embodiments, the combination product is used for the prevention or treatment of tumors. In some embodiments, the tumor is cancer, etc. In some embodiments, the combination product is used for the prevention or treatment of infections, such as chronic infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, etc.

[0243] In some regimens, two or more components of the combined product may be administered to the subject sequentially, separately, or simultaneously.

[0244] In some embodiments, the present invention also provides a kit comprising the CTLA-4 binding molecule, pharmaceutical composition, immunoconjugate, or combination product of the present invention, and optionally a package insert with instructions for administration.

[0245] In some embodiments, the present invention also provides pharmaceutical articles comprising the CTLA-4 binding molecule of the present invention, pharmaceutical compositions, immunoconjugates, and combination products, optionally including a packaging insert for instructions of administration.

[0246] IX. Use of the CTLA-4 binding molecule of the present invention

[0247] In one aspect, the present invention relates to a method for modulating an immune response in an individual. The method comprises administering to a subject an effective amount of the CTLA-4 binding molecule disclosed herein, or a pharmaceutical composition, immunoconjugate, or combination product comprising said CTLA-4 binding molecule, thereby modulating an immune response in the subject. In one embodiment, the therapeutically effective amount of the CTLA-4 binding molecule, pharmaceutical composition, immunoconjugate, or combination product disclosed herein restores, enhances, stimulates, or increases an immune response in a subject.

[0248] In some embodiments, the present invention relates to a method for inhibiting the activity of CTLA-4 in an individual, blocking the binding of CTLA-4 to CD80 molecules, blocking the binding of CTLA-4 to CD86 molecules, and inducing the secretion of IL-2 and / or TNF-α, the method comprising administering to a subject an effective amount of the CTLA-4 binding molecule disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising thereto.

[0249] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, said method comprising administering to said subject an effective amount of the CTLA-4 binding molecule disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising therein. In some embodiments, the tumor is a tumor immune escape. In some embodiments, said tumor is cancer.

[0250] In another aspect, the present invention relates to a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the CTLA-4 binding molecule disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising therein. In one embodiment, the infectious disease is a chronic infection, such as tuberculosis.

[0251] In another aspect, the present invention relates to a method for inducing antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of the CTLA-4 binding molecule disclosed herein or a pharmaceutical composition or immunoconjugate or combination product comprising therein.

[0252] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient with or at risk of having the diseases described herein). In one embodiment, the subject has or is at risk of having the diseases described herein (e.g., a tumor or infectious disease as described herein). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. Alternatively or in combination, the subject is immunocompromised due to an infection or is at risk of being immunocompromised due to an infection.

[0253] In some implementations, the tumors described herein, such as cancers, include, but are not limited to, solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions.

[0254] Examples of solid tumors include malignant tumors, such as sarcomas and carcinomas (including adenocarcinoma and squamous cell carcinoma) affecting multiple organ systems, such as those invading the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidney, bladder epithelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Squamous cell carcinomas include malignant tumors such as those in the lungs, esophagus, skin, head and neck region, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, for example, advanced melanoma. In one embodiment, the cancer is renal cell carcinoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention.

[0255] 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), such as 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 IV melanoma) or Merkel cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), spinal dysplasia syndrome, bladder cancer (e.g., transitional cell carcinoma), kidney cancer (e.g., renal cell carcinoma, such as clear cell renal cell carcinoma, such as advanced or metastatic clear cell renal cell carcinoma), and colon cancer. In addition, refractory or recurrent malignant tumors can be treated with the CTLA-4 binding molecules described herein or pharmaceutical compositions or immunoconjugates or combination products containing them.

[0256] In one implementation, the disease is one with elevated levels of CTLA-4 (nucleic acid or protein). In some implementations, the tumor is one that can be suppressed by inhibiting the binding of CTLA-4 to CD80 and / or CD86 molecules, such as cancer. In some implementations, the tumor or infection is a disease that would benefit from suppressing CTLA-4 levels of nucleic acid or protein.

[0257] In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is persistent, latent, or slow-growing. In some embodiments, the chronic infection is caused by a pathogen selected from bacteria, viruses, fungi, and protozoa.

[0258] In another embodiment, the chronic infection is tuberculosis caused by Mycobacterium tuberculosis (MTB), which can affect many organs, with pulmonary tuberculosis being the most common. The CTLA-4 binding molecule can be combined with conventional tuberculosis treatment for therapeutic advantages. In some embodiments, the CTLA-4 binding molecule is administered in combination with drugs such as isoniazid, rifampin, streptomycin, pyrazinamide, ethambutol, or thiourea.

[0259] In some embodiments, the CTLA-4 binding molecule of the present invention or an immune conjugate or composition or combination product comprising it may delay the onset of symptoms and / or symptoms associated with the condition.

[0260] In some embodiments, the preventive or treatment methods described herein also include administration to the subject or individual in combination of the CTLA-4 binding molecule or pharmaceutical composition or immunoconjugate or combination product disclosed herein, and one or more other therapies, such as modes of treatment and / or other therapeutic agents.

[0261] In some implementations, the treatment modality includes surgery (e.g., tumor resection); radiotherapy (e.g., external beam therapy, which involves three-dimensional conformal radiotherapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed at a pre-selected target or organ), or focused irradiation, etc. Focused irradiation can be selected from stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy. Focused irradiation can have a radiation source selected from particle beams (protons), cobalt-60 (photons), and linear accelerators (X-rays), for example, as described in WO 2012 / 177624.

[0262] Radiation therapy can be administered through one or a combination of several methods, including but not limited to external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, whole-body radiation therapy, radiotherapy, and permanent or transient interstitial brachytherapy.

[0263] In some implementations, the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

[0264] Other exemplary 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., agonistic GITR antibodies or CD137 antibodies), etc. Preferably, the other antibodies are selected from anti-PD-1 antibodies and / or anti-PD-L1 antibodies. More preferably, the anti-PD-1 antibody is nivolumab from BMS or pembrolizumab from Merck; the anti-PD-L1 antibody is atezolizumab from Roche, avelumab co-developed by Merck KGaA and Pfizer, or durvalumab from AstraZeneca.

[0265] In some embodiments, the immunomodulator is an activator or agonist of the co-stimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from agonists of the following molecules (e.g., agonistic antibodies or their antigen-binding fragments, or soluble fusions): OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligands.

[0266] The combination therapies of the present invention encompass both combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration. In the case of separate administration, the CTLA-4 binding molecule or immune conjugate of the present invention may be administered before, simultaneously with, and / or after the administration of other therapies.

[0267] In one implementation, the administration of the CTLA-4 binding molecule and the administration of other therapies (e.g., treatment modalities or therapeutic agents) occur within each other over a period of about one month, or about one, two, or three weeks, or about one, two, three, four, five, or six days.

[0268] The CTLA-4 binding molecule of the present invention (and pharmaceutical compositions or immunoconjugates comprising it) can be administered by any suitable method, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if local treatment requires, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it can be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including but not limited to single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0269] For the prevention or treatment of disease, the appropriate dosage of the CTLA-4 binding molecule of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of CTLA-4 binding molecule, the severity and progression of the disease, whether the CTLA-4 binding molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the CTLA-4 binding molecule, and the judgment of the attending physician. The CTLA-4 binding molecule is appropriately administered to the patient as a single treatment or in the course of a series of treatments. The dosage and treatment regimen of the CTLA-4 binding molecule can be determined by a skilled technician.

[0270] It is understood that the immune conjugates or compositions or combination products of the present invention can be used to replace the CTLA-4 binding molecules for any of the above-described prevention or treatment.

[0271] X. Methods and compositions for diagnosis and detection

[0272] In some embodiments, any CTLA-4 binding molecule provided herein can be used to detect the presence of CTLA-4 in a biological sample. The term "detection," as used herein, includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, or ELISA assays. In some embodiments, the biological sample is blood, serum, or other bodily fluid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from hyperplastic or cancerous lesions.

[0273] In one embodiment, a CTLA-4 binding molecule is provided for use in a diagnostic or detection method. In another aspect, a method for detecting the presence of CTLA-4 in a biological sample is provided. In some embodiments, the method includes detecting the presence of the CTLA-4 protein in the biological sample. In some embodiments, CTLA-4 is human CTLA-4. In some embodiments, the method includes contacting the biological sample with a CTLA-4 binding molecule as described herein under conditions that allow the CTLA-4 binding molecule to bind to CTLA-4, and detecting whether a complex is formed between the CTLA-4 binding molecule and CTLA-4. The formation of the complex indicates the presence of CTLA-4. This method can be in vitro or in vivo. In one embodiment, the CTLA-4 binding molecule is used to select subjects suitable for treatment with the CTLA-4 binding molecule, for example, where CTLA-4 is a biomarker used for selecting said subjects.

[0274] In one embodiment, the CTLA-4 binding molecule of the present invention can be used to diagnose cancer or tumors, such as to evaluate (e.g., monitor) the treatment or progression of the disease described herein (e.g., hyperplastic or cancerous disease), its diagnosis, and / or staging. In some embodiments, a labeled CTLA-4 binding molecule is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C 125 I, 3 H and 131 I. Fluoresceins such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labeling, phage labeling, stable free radicals, etc.

[0275] In some embodiments of any of the inventions provided herein, the sample is obtained prior to CTLA-4-based molecular therapy. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin-fixed and paraffin-coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., from a surgically removed specimen), or a fine-needle aspirate.

[0276] In some implementations, CTLA-4 is tested before treatment, for example, before initiating treatment or before a treatment after a treatment interval.

[0277] In some embodiments, a method for treating a tumor or infection is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample containing cancer cells) for the presence of CTLA-4, thereby determining a CTLA-4 value; comparing the CTLA-4 value with a control value (e.g., the value of CTLA-4 in a sample from a healthy individual); and if the CTLA-4 value is greater than the control value, administering to the subject a therapeutically effective amount of a CTLA-4 binding molecule (e.g., the CTLA-4 binding molecule described herein) optionally in combination with one or more other therapies, thereby treating the tumor or infection.

[0278] It is understood that the various embodiments described in the various parts of this invention, such as diseases, therapeutic agents, treatment methods, and administration, are also applicable to embodiments in other parts of this invention, or can be combined with embodiments in other parts. The embodiments described in the various parts of this invention, such as the properties, uses, and methods applicable to CTLA-4 binding molecules, are also applicable to compositions, conjugates, combination products, and kits containing CTLA-4 binding molecules.

[0279] Example

[0280] Example 1. Animal Immunity and Serum Titer Detection

[0281] I. Animal Immunization

[0282] Human CTLA-4 protein (GenBank ID: NP_005205.2) (purchased from Beijing Sinocare Biotechnology Co., Ltd., catalog number: 11159-HNAH) was used as an antigen for immunization of alpacas. The human CTLA-4 protein was prepared with an adjuvant before administration to the alpacas.

[0283] Specifically, 500 μg of human CTLA-4 protein was prepared into an emulsion with Freund's complete adjuvant (for primary immunization) or with Freund's incomplete adjuvant (for booster immunization) and administered to alpacas every 2 weeks for a total of 4 immunizations.

[0284] II. Serum titer testing

[0285] Two weeks after the last immunization of the alpacas, blood was collected from the alpacas. The serum was analyzed using ELISA to determine the binding capacity of the alpaca serum to the human CTLA-4 protein after immunization. The antibody titer in the serum was used to assess the effectiveness of the antigen immunization. The specific method is as follows:

[0286] II.1 Antigen Coating: One day before the antibody titer in serum was measured post-immunization, human CTLA-4 protein (purchased from Beijing Sinocare Biotechnology Co., Ltd., catalog number: 11159-HNAH), used as the antigen, was diluted with PBS buffer to a final concentration of 2 μg / mL to obtain the antigen dilution. This dilution was added to each well of a 96-well plate at a rate of 30 μL / well, and the plate was incubated overnight at 4°C. On the day of the immunotiter assay, the 96-well plate was washed three times with PBS, then blocked with PBST containing 5% skim milk powder at room temperature for two hours, followed by three washes with PBS buffer.

[0287] II.2 Serum dilution: On another 96-well plate, negative alpaca serum that had not been immunized with human CTLA-4 protein and alpaca serum that had been immunized with human CTLA-4 protein were serially diluted with PBS. The first well was a 200-fold dilution, and the subsequent 7 wells were serially diluted 2-fold based on the first well.

[0288] II.3 Antigen-antibody binding reaction: Add 30 μL / well of the diluted serum from II.2 to each well of the antigen-coated plate prepared in II.1, and incubate at 37°C for 1 hour. Discard the supernatant, wash the plate twice with PBS buffer, and then add 30 μL of a 1:5000 diluted second antibody (MonoRab) to each well. TM Rabbit anti-cameloid VHH antibody (GenScript Biotechnology Co., Ltd., catalog number: A01862-200), incubated at 37℃ for 0.5h.

[0289] II.4 Colorimetric Reading: Discard the supernatant, wash each well of the 96-well plate three times with PBS, then add 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) substrate for color development for 5 minutes. Terminate the reaction with H2SO4, and measure the optical density (OD) at 450 nm using an ELISA plate reader (Molecular Devices, SpecterMax 190). The results are shown in Table 2.

[0290] Table 2. Results of ELISA colorimetric assays on alpaca serum diluted at different ratios.

[0291] serum dilution factor <![CDATA[OD of negative serum 450nm > <![CDATA[OD of NSY007 alpaca serum 450nm > 1:200 0.348 2.268 1:400 0.219 2.306 1:800 0.186 1.93 1:1600 0.147 1.395 1:3200 0.122 1.188 1:6400 0.103 0.788 1:12800 0.071 0.479 1:25600 0.09 0.335

[0292] Note: Negative serum is alpaca serum that has not been immunized with human CTLA-4 protein.

[0293] As shown in Table 2 above, the NSY007 alpaca showed good immunization efficacy after immunization with human CTLA-4 protein. This NSY007 alpaca was then used for the construction of a peripheral blood antibody library.

[0294] Example 2. Construction of a cDNA gene library of VHH antibody

[0295] As described in Example 1, two weeks after the last immunization of the alpacas, 50 mL of fresh blood was collected from NSY007 alpacas, which showed good immunization efficacy in the ELISA experiment. Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Paque density gradient separation solution for the construction of a VHH antibody cDNA gene library. The specific method is as follows:

[0296] I. Isolation of peripheral blood mononuclear cells

[0297] 15 mL of Ficoll-Paque density gradient separation buffer (purchased from GE, catalog number: 17144003S) was slowly added to a 50 mL centrifuge tube. 50 mL of collected NSY007 alpaca blood was anticoagulated with heparin, then diluted with PBS buffer at a 1:1 volume ratio. The centrifuge tube was tilted, and 30 mL of the alpaca blood PBS dilution was slowly added along the tube wall to maintain a clear separation interface between the Ficoll-Paque density gradient separation buffer and the alpaca blood PBS dilution. The 50 mL centrifuge tube containing the blood and separation buffer was centrifuged at 4°C for 20 min, with the centrifuge set to 1500 rpm and an acceleration of 3°C / 0°C. After centrifugation, the liquid surface separated into four layers: the upper layer was a plasma mixture, the lower layer consisted of erythrocytes and granulocytes, the middle layer was Ficoll-Paque PLUS, and at the boundary between the upper and middle layers was a narrow white band dominated by PBMCs, i.e., the PBMC cell layer. Carefully use a pipette to aspirate the PBMC cells from the center and transfer them to a new 50 mL centrifuge tube. Wash the PBMC cells twice with PBS buffer, centrifuging at 1500 rpm for 10 min at 4°C after each wash. Finally, collect the PBMC cell pellet, resuspend it in 1.5 mL of PBS buffer, and count the cells under a microscope.

[0298] II. Construction of VHH antibody cDNA gene library

[0299] Total RNA was extracted from PBMCs isolated using standard methods. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, catalog number: 6210A).

[0300] Since the molecular form of antibodies in alpaca blood is different from that of ordinary antibodies, they do not contain light chains and heavy chains do not contain CH1. Therefore, primer pairs were designed at the N-terminus and CH2 of VHH to perform PCR to obtain two fragments of different sizes. The smaller target fragment was then recovered by gel extraction to remove the large VH gene fragment contained in the PCR product, thus obtaining a small fragment PCR amplification product.

[0301] By comparing the amino acid sequences of the VHH gene, which is common in all alpaca lineages, degenerate primers specific to the VHH gene of alpaca lineages were designed, with NcoI and NotI restriction enzyme sites at both ends, respectively.

[0302] Using the recovered small-fragment PCR amplification products as templates, all VHH genes were amplified using degenerate primers specific to the alpaca strain VHH gene. The primers used were derived from the literature by Sabir JSM et al., Construction of The primers used in camelidsVHH repertoire in phage display-based library, CRBiologics, 2014, 337(4):244-249 were modified by replacing the restriction enzyme sites of the primers in the literature with NotI and NcoI. Two rounds of PCR amplification were performed, and the PCR amplification conditions are shown in the table below.

[0303] Table 3. One-round PCR amplification reaction system

[0304]

[0305]

[0306] Table 4. Procedure for one round of PCR amplification

[0307]

[0308] A total of 32 reaction systems were performed in one round of PCR, and the amplicon was purified, with a total of 3.6 μg of product recovered.

[0309] The products from the first round of PCR were used as templates for a second round of PCR amplification. The PCR system and procedure are as follows:

[0310] Table 5. Second-round PCR amplification reaction system

[0311] Components of the reaction system The amount required for a single reaction system 2×PrimeSTAR Max DNA Polymerase 25μL Primer-F 1μL Primer-R 1μL template 10ng <![CDATA[ddH2O]]> Make up to 50 μL

[0312] Table 6. Two-round PCR amplification reaction procedure

[0313]

[0314] A total of 96 reaction systems were performed in the second round of PCR, and the amplicon was purified, with a total product recovery of 13.82 μg.

[0315] The pHEN1 phage surface display vector (4539 bp) was double-digested with restriction enzymes NcoI and NotI (obtained from BioVector NTCC Inc.).

[0316] Table 7. Vector Enzyme Digestion System

[0317] Components of the reaction system Amount required for one reaction system carrier pHEN1 1.0μg Restriction enzyme NotI 0.6μL restriction enzyme NcoI 1.0μL 10×FD Green Buffer 2.0μL <![CDATA[ddH2O]]> Bring the volume to 20.0 μL.

[0318] Similarly, all VHH antibody genes were recovered after double digestion with restriction enzymes NcoI and NotI followed by two rounds of PCR amplification.

[0319] The pHEN1 phage surface display vector treated with restriction enzymes was mixed with the VHH antibody gene treated with restriction enzymes at a mass ratio of 2.5:1. Ligase (purchased from Takara, catalog number: 2011A) was added to the mixture. The resulting mixture was incubated at 16°C for two hours. In this manner, the VHH antibody gene was ligated into the pHEN1 phage surface display vector, such that the C-terminus of the VHH antibody gene was fused to the N-terminus of the GIII gene in the phage expression vector. Specifically, following the instructions of the Takara ligase kit and considering the target library size, a sufficient volume of ligation reaction was performed. The ligation system was designed as follows (total volume of a single reaction was 20 μL):

[0320] Table 8. Connection Reaction System

[0321]

[0322] The ligation products were recovered using a recovery kit (Omega, catalog number: D6492-02). The ligation products were then transformed into competent Escherichia coli SS320 (Lucigen, catalog number: MC1061 F) by electroporation using a Bio-Rad, MicroPulser electroporator.

[0323] 10 μL of transformed *E. coli* SS320 bacterial culture was serially diluted 10-fold with antibiotic-free 2YT medium. 2 μL of each dilution was plated onto 2YT plates containing 100 μg / mL carbenicillin. The total number of colonies formed by electrotransformation was calculated, i.e., the library capacity, by counting the colonies formed on the plates. The calculated capacity of this immunotherapy library was 1 × 10⁻⁶. 9 One VHH antibody gene (cfu / mL). The bacterial count corresponding to the library volume is 2 OD. 600 That is, the OD of 1 mL of antibody library 600 The value is 2OD 600 .

[0324] Example 3. Demonstrates the preparation of phage libraries for antibodies and the screening of antibody candidate molecules.

[0325] In this embodiment, a phage library displaying VHH antibodies against human CTLA-4 was first prepared. Then, the phage library was screened using different methods, and the affinity of the selected candidate VHH antibodies for binding to the antigen was analyzed.

[0326] I. Preparation of phage libraries displaying antibodies

[0327] Take a volume of bacteria 10 times that of Example 2 (approximately 20 OD). 600 1×10 10 1 ml of the transformed *E. coli* (1000 cells) was diluted with fresh 2-YT liquid medium, adjusting the amount of 2-YT medium added to achieve an initial OD value of approximately 0.05. The diluted bacterial suspension was incubated at 37°C and 220 rpm until the logarithmic growth phase. At this point, VCSM13 helper phage (purchased from Stratagene) was added at a concentration 50 times the bacterial count (i.e., a multiplicity of infection (MOI) of approximately 50), thoroughly mixed, and allowed to stand for 30 min. The suspension was then incubated for 1 h with shaking at 220 rpm. After centrifugation at 10000 rpm for 5 min, the culture medium was replaced with 2-YT medium containing both carbenicillin (50 μg / mL) and kanamycin (40 μg / mL), and the suspension was incubated overnight at 30°C and 220 rpm with shaking. The next day, the bacterial culture was centrifuged at 13000g for 10 min, and the supernatant was precipitated by adding 20% ​​PEG / NaCl solution to obtain a phage library displaying VHH antibodies against human CTLA-4. After washing once with PBS, it was used to screen for VHH antibodies that could specifically bind to CTLA-4.

[0328] II. Screening of antibody candidate molecules

[0329] II.1 Screening of phage libraries using magnetic bead method

[0330] Magnetic bead screening of phage libraries is based on the process of binding biotin-labeled recombinant human CTLA-4 protein to anti-biotin-conjugated magnetic beads for screening antibody phage libraries. First, biotin-labeled recombinant human CTLA-4 protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11159-HNAH) was used. The biotin-labeled CTLA-4 protein and Dynabeads (purchased from Invitrogen) were incubated under the Kingfisher magnetic bead screening system method, with the beads bound and washed using PBS containing 5% bovine serum albumin (BSA) as the blocking solution. This ensured the biotin-labeled CTLA-4 protein bound to the magnetic beads. The magnetic beads bound to the CTLA-4 antigen and the aforementioned phage library displaying nanobodies were incubated at room temperature for 2 hours. After washing 6-8 times with PBST buffer, non-specifically adsorbed phages were removed. Add trypsin (purchased from Gibco, catalog number 25200072) and mix gently for 20 min to elute the nanobody displaying the phage that specifically binds to human CTLA-4 protein.

[0331] Next, the eluted phages were amplified. Specifically, the eluted phages were used to infect logarithmic-phase SS320 cells (purchased from Lucigen, catalog number: MC1061 F), and the phage-infected SS320 cells were plated on 2YT agar plates containing 50 μg / mL carbenicillin and incubated overnight at 37°C. The phage library was collected the next day. Other steps in preparing the phage library using SS320 cells are described in Example 3.I. The obtained phage library was then used for a second round of screening. Specifically, magnetic beads bound to CTLA-4 protein and the phage library were incubated at room temperature for 2 hours. After washing 6-8 times with PBST buffer to remove non-specifically adsorbed phages, and removing residual liquid, only the magnetic beads remained. CD80 protein solution was added to the magnetic beads for competitive binding, removing phages with weaker binding affinity to CTLA-4 protein. After washing 6-8 times with PBST buffer, non-specifically adsorbed phages were removed. The protein sequence of the CTLA-4 ligand protein CD80 used is NP_005182.1, which can be purchased from Sinocare Pharmaceuticals, catalog number 10698-HCCH.

[0332] Diluted trypsin (purchased from Gibco, catalog number 25200072) was added to the magnetic beads eluted with PBST, and the reaction was carried out for 30 min. The supernatant was then centrifuged to obtain phage particles.

[0333] The phages obtained in the second round of amplification / binding screening were also used in the third round of amplification / binding screening, eluted using various methods including CTLA-4 ligand protein CD80 competition and trypsin. This enriched VHH display phages that specifically bind to human CTLA-4 protein. This process was repeated, with 10 clones randomly selected in each round for sequence analysis.

[0334] Sequencing was performed on 111 monoclonal phages obtained after three rounds of screening. The results showed that there were partial repetitions in the gene sequences of different clones, indicating that the magnetic bead method for screening phage libraries can significantly enrich VHH antibody sequences.

[0335] II.2 Immunotube method for screening phage libraries

[0336] Immunotube screening is based on coating the surface of immunotubes with antigens and screening for antibody-displaying phages that specifically bind to the target antigen. Specifically, the day before screening, 2 ml of recombinant human CTLA-4 protein solution (purchased from Corning) is injected into immunotubes (purchased from Corning) and left to stand overnight, thereby coating the immunotubes with recombinant human CTLA-4 protein. The immunotubes coated with human CTLA-4 antigen and the phage library displaying nanobodies are incubated at room temperature for 2 hours. After washing 6-8 times with PBST, non-specifically adsorbed phages are removed. Trypsin (purchased from Gibco, catalog number 25200072) is added and gently mixed for 20 min to elute the nanobodies displaying phages that specifically bind to human CTLA-4 protein.

[0337] Next, the eluted phages were amplified. Specifically, the eluted phages were used to infect logarithmic-phase SS320 cells (purchased from Lucigen, catalog number: MC1061 F), and the phage-infected SS320 cells were plated on 2YT plates containing 50 μg / mL carbenicillin and incubated overnight at 37°C. The phage library was collected the next day. Other steps for preparing the phage library using SS320 cells are described in Example 3.I. The obtained phage library was then used for a second round of screening. Immunotubes coated with human CTLA-4 antigen and the phage library were incubated at room temperature for 2 hours. After washing 6-8 times with PBST, non-specifically adsorbed phages were removed. In the second round of screening, elution was performed using multiple methods, including CTLA-4 ligand protein CD80 competition and trypsin. The phages obtained in the second round of screening were used in the third round of screening. In the third round, elution was performed using CTLA-4 ligand protein CD80 competition and trypsin. This process was repeated, with 10 clones randomly selected from each round for sequence analysis.

[0338] The 124 monoclonal phages obtained after three rounds of screening were amplified and sequenced in SS320 cells. The results showed that there were partial repetitions in the gene sequences of different clones, indicating that the immunotube method can significantly enrich the sequences of phage libraries.

[0339] III. Activity identification of candidate molecules

[0340] III.1 Preparation of Monoclonal Lysis Buffer for Antibody VHH Molecules

[0341] The bacterial culture of the monoclonal bacteria obtained in Example 3.II was inoculated at a ratio of 1:1000 into 50 mL of double antibiotic medium containing 50 μg / mL carbenicillin and 12 μg / mL tetracycline. The culture was incubated at 37°C with shaking for 14 h. After centrifugation at 10000g for 5 min, the bacteria were resuspended in 1 mL of Tris-HCl buffer (pH 9.0) containing Benzonase nuclease (Merck, 70746-3) (Benzonase nuclease concentration of 0.002 U / mL). The cells were lysed on ice for 30 min and centrifuged at 10000g for 10 min at 4°C. The supernatant was collected to obtain a monoclonal cell lysate containing the antibody VHH molecule.

[0342] III.2 Determining the affinity of candidate molecules by flow cytometry

[0343] i) Construction and identification of cell lines stably overexpressing CTLA-4

[0344] CHO-s cell lines that stably overexpress full-length human CTLA-4 (GenBank GI number: NP_005205.2) and rhesus monkey CTLA-4 (GenBank GI number: XM_015110722.2) were constructed, and the overexpressing cell lines were identified using FACS.

[0345] 1. Construction of stable overexpression cell lines

[0346] 1.1 Construction of plasmid expressing human CTLA-4 extracellular domain protein

[0347] Protein expression vectors were constructed using the synthesized gene sequences. The plasmid construction method for the expression vectors was as follows: DNA fragments containing the full-length protein gene sequences of human CTLA-4 and rhesus monkey CTLA-4 were synthesized using gene synthesis methods. These two synthesized gene fragments and the expression vector pcDNA3.3-TOPO (Invitrogen product number: K830001) were digested with restriction enzymes HindIII and SmallI and then ligated. The plasmids were introduced into SS320 E. coli, and single colonies were picked and sequenced to obtain the correct plasmid clones. Plasmid extraction and resequencing were performed to confirm the correct plasmid construction.

[0348] 1.2 Electroconversion

[0349] Chinese hamster ovarian cancer cells (CHO-s) (Invitrogen) were cultured in CD-CHO serum-free medium (purchased from Gibco, catalog number: 10743029). Cells were passaged to 5 × 10⁶ cells / day before electroporation. 6 Cells / mL, use the electroporation kit (Invitrogen, Neon) on the same day of electroporation. TM The constructed plasmids were introduced into CHO-s cells using a kit (MPK10096) and an electroporator (Invitrogen, Neon™ Transfection System, MP922947) at 1700V / 20ms / 1 cycle. The electroporated cells were then added to 3 mL of CD-CHO medium and incubated at 37°C in a CO2 incubator for 48 hours.

[0350] 1.3 Cell Plating and Culture

[0351] After electroporation and culture in section 1.2, CHO-s cells were seeded at a rate of 2000 cells / well into 96-well plates. A final concentration of 30 μM methionine imino sulfone (MSX) (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C-100 ml) were added, and the plates were incubated in a 37°C CO2 incubator. After 10 days, medium containing 30 μM MSX and 1×GS supplement was added.

[0352] 1.4 Clonal Identification and Cell Expansion Culture

[0353] The grown clones were selected and transferred to 24-well cell culture plates for culture. CHO-s cells that overexpressed human CTLA-4 (hereinafter referred to as human CTLA-4-CHO cells) and CHO-s cells that overexpressed rhesus monkey CTLA-4 (hereinafter referred to as rhesus monkey CTLA-4-CHO cells) were obtained, respectively.

[0354] ii) Obtaining or preparing control antibodies

[0355] Ipilimumab was used as a control antibody. This control antibody can be purchased from Bristol-Myers Squibb (BMS), the manufacturer of the ipilimumab antigen, or it can be prepared as follows:

[0356] The entire light and heavy chain genomes of the control antibody were expressed using the ExpiCHO transient transduction expression system. The culture medium was ExpiCHO. TMExpression Medium (Gibco, A29100-01), transfection kit: ExpiFectamine TM CHOTransfection Kit (Gibco, A29129). The specific method is as follows: The complete light chain gene sequence (nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 34) and the complete heavy chain gene sequence (nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 35) of the control antibody - ipilimumab (BMS) were synthesized separately (gene synthesis supplier: Universal Biotechnology). ExpiCHO expression plasmids containing the ipilimumab antibody light and heavy chain genes were constructed by molecular cloning, and the plasmids containing the ipilimumab antibody light and heavy chain genes were mixed at a mass ratio of 2:1.

[0357] One day before transfection, ExpiCHO cells were passaged. Following the transfection kit instructions, 25 μg of the constructed plasmid mixture was mixed with the transfection reagent in a 25 mL system. This mixture was then added dropwise to 25 mL of ExpiCHO cell culture and thoroughly mixed. The cells were then incubated at 37°C for 18-22 hours. GS supplement (Sigma, 58672C-100 mL) was added to the transfection mixture. After supplementation, the cells were incubated at 32°C. On day 5 post-transfection, a second supplementation was added, and the cells were incubated at 32°C for 10-12 days. The expressed cell suspension was then centrifuged at 12000-15000g for 10-30 minutes. The supernatant was collected, filtered through a 0.22μm filter, and purified using Protein A / G affinity chromatography. The target protein was eluted with 100mM glycine buffer (pH 3.0), followed by neutralization to pH 7.0 with 1M Tris-HCl buffer. Small samples were taken, identified by SDS-PAGE, aliquoted, and stored frozen. The antibody concentration was 1.46 mg / ml.

[0358] iii) Affinity determination of candidate molecules

[0359] First, collect cultured human CTLA-4-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in prepared FACS buffer, count them, and adjust the cell suspension density to 2×10⁻⁶. 6Human CTLA-4-CHO cells were added to 100 μL per well in a 96-well round-bottom plate and centrifuged at 300 g to remove the supernatant. Serially diluted antibody (prepared in section III.1) and control antibody (15 μg / mL) were added to the corresponding wells at concentrations of 1:1, 1:3, and 1:9, or at concentrations of 1:1, 1:6, 1:36, and 1:216. Cells were homogenized using a multipipe and incubated at 4°C for 30 minutes. The cell mixture was centrifuged at 300 g to remove the supernatant. 200 μL of FACS buffer was added to the corresponding wells and the cells were resuspended using a multipipe. This washing step was repeated twice, and the cells were centrifuged at 300 g to remove the supernatant. PE-labeled anti-human IgG Fc flow cytometry antibody (Abcam, 98596) was added, and the cells were homogenized using a multipipe and incubated at 4°C for 30 minutes. 200 μL of the mixture was centrifuged at 300 g to remove the supernatant. Resuspend the cells in FACS buffer; repeat this washing step twice, add 200 μL of FACS buffer to each well, resuspend the cells, and detect the fluorescence intensity using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). A negative control (NC) was set up, consisting of purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502), abbreviated as "isotype control" in the figure.

[0360] The results are as follows Figure 1A , Figure 1B and Figure 1C As shown, preliminary screening using the FACS method identified proteins containing antibodies with specific binding activity to CTLA-4 in the antibody lysate. These ten candidate molecules, with clone numbers NB25gb-21, NB25ga-14, NB25B-17, NB25gb-6, NB25gb-1, SY23-32, SY23-33, SY23-119, SY23-146, and SY23-162, exhibited good affinity.

[0361] Example 4. Expression and Identification of Chimeric VHH-Fc Antibody

[0362] In this embodiment, the expression and analysis of VHH-Fc chimeric antibody protein fused with the Fc fragment were performed.

[0363] First, the expression vector was constructed. Ten positive VHH candidate antibodies obtained through screening in Example 3 were fused with the human IgG1 Fc fragment. The positive VHH gene sequence was linked to the C-terminus of the human IgG1 Fc gene sequence to the N-terminus, and then ligated into the expression vector pcDNA3.3-TOPO (Invitrogen product number: K830001). This expression vector with the VHH-Fc sequence was transformed into ExpiCHO cells, and expression of the VHH-Fc chimeric antibody protein fused with the Fc fragment was induced.

[0364] Antibody expression was performed using the ExpiCHO transient expression system, and the culture medium was ExpiCHO. TM ExpressionMedium (Gibco, A29100-01), transfection kit: ExpiFectamine TM CHO Transfection Kit (Gibco, A29129). The specific method is as follows: One day before transfection, ExpiCHO cells were passaged. In a 25 mL system, 25 μg of the constructed fusion expression vector was mixed with the transfection reagent and then added dropwise to 25 mL of ExpiCHO cells. The mixture was thoroughly mixed and incubated at 37°C for 18-22 hours. Then, according to the kit instructions, GS supplement (Sigma, 58672C-100 mL) was added, and the cells were incubated at 32°C. On day 5 post-transfection, a second supplement was added, and the cells were incubated at 32°C. After 10-12 days, the expressed cell suspension was centrifuged at 12000-15000g for 10-30 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter and purified using the Protein A / G affinity purification method. The target protein was eluted with 100 mM glycine (pH 3.0) and then neutralized with 1 M Tris-HCl. The concentration of each VHH-Fc chimeric antibody protein was 500 μg / mL, and it was stored at 4℃ for later use.

[0365] Example 5. In vitro detection of the binding activity of VHH-Fc candidate antibody to human CTLA-4 protein on cells.

[0366] The VHH-Fc candidate antibodies obtained in Example 4 were evaluated. The in vitro binding activity of each VHH-Fc candidate antibody to CTLA-4 protein on cells was detected by FACS method. The specific method is as follows.

[0367] 1) Collect cultured human CTLA-4-CHO cells, centrifuge at 300g to remove supernatant, resuspend cells in prepared FACS buffer, count cells, and adjust the cell suspension density to 2×10⁻⁶. 6 Cells / mL.

[0368] 2) Add 100 μL of human CTLA-4-CHO cells to each well of a 96-well round-bottom plate and centrifuge at 300g to remove the supernatant;

[0369] 3) Add 100 μL of candidate antibody dilution buffer, serially diluted with FACS buffer, to the corresponding wells at the following concentrations: Figure 2A , Figure 2B As shown, control antibody dilution solution was prepared, cells were homogenized using a pipette and incubated at 4°C for 30 minutes;

[0370] 4) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200μL of FACS buffer to the corresponding well and resuspend the cells using a pipette;

[0371] 5) Repeat step 4) twice, centrifuge at 300g and discard the supernatant;

[0372] 6) Add PE-labeled anti-human IgG Fc flow cytometry antibody (Abcam, ab98596), mix the cells with a pipette, and incubate at 4°C for 30 minutes;

[0373] 7) Centrifuge at 300g to remove supernatant, add FACS buffer and resuspend cells;

[0374] 8) Repeat step 7) twice, add 200 μL of FACS buffer to each well, resuspend the cells, and detect the fluorescence intensity using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0375] like Figure 2A and Figure 2B As shown in the FACS experiment, the eight VHH molecules screened in Example 3 were fused with Fc to obtain VHH-Fc chimeric antibodies with high affinity, and the affinity was comparable to or better than that of the control antibody IPI.

[0376] Example 6. Chimeric VHH-Fc antibody blocks the binding of CTLA-4 to CD80 / CD86.

[0377] The obtained VHH-Fc candidate antibodies were evaluated, and their blocking activity against CTLA-4 binding to CD80 / CD86 was detected using the FACS method, as follows:

[0378] 1) Collect cultured human CTLA-4-CHO cells, centrifuge at 300g to remove supernatant, resuspend cells in prepared FACS buffer, count cells, and adjust the cell suspension density to 2×10⁻⁶. 6 Cells / mL.

[0379] 2) Add 100 μL of human CTLA-4-CHO cell suspension to each well of a 96-well round-bottom plate, centrifuge at 300g to remove the supernatant;

[0380] 3) Add serially diluted candidate antibody and control antibody dilution to the corresponding wells, blow the cells evenly with a pipette and incubate at 4°C for 30 minutes;

[0381] 4) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200μL of FACS buffer to the corresponding well and resuspend the cells using a pipette;

[0382] 5) Repeat step 4) twice, centrifuge at 300g and discard the supernatant;

[0383] 6) Add 100 μL of huCD80_EDC mFc (storage concentration of 98.55 μg / mL) or huCD86_EDC-mFc (storage concentration of 112.07 μg / mL) diluent to the corresponding well. The concentration after dilution is 0.0625 μg / mL.

[0384] 7) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 100μL of diluted CTLA-4 antibody dilution solution to the corresponding well, resuspend the cells and incubate them at 4℃ for 30 minutes;

[0385] 8) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200μL of FACS buffer to the corresponding well and resuspend the cells using a pipette;

[0386] 9) Repeat step 8) twice, centrifuge at 300g and discard the supernatant;

[0387] 10) Add PE-labeled anti-human Fc antibody (eBioscience, 98742), mix the cells with a pipette and incubate at 4°C for 30 minutes;

[0388] 11) Centrifuge at 300g to remove supernatant, add FACS buffer and resuspend cells;

[0389] 12) Repeat step 11) twice, add 200 μL of FACS buffer to each well, resuspend the cells, and detect them by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0390] like Figure 3A and Figure 3B As shown in the FACS experiment, the VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc have high blocking activity against the binding of CTLA-4 and CD80, and the blocking activity is similar to that of the control antibody IPI.

[0391] like Figure 4A and Figure 4B As shown in the FACS experiment, the VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc have high blocking activity against the binding of CTLA-4 and CD86, and the blocking activity is similar to that of the control antibody IPI.

[0392] Example 7. Activity of chimeric VHH-Fc antibody binding to monkey CTLA-4

[0393] To evaluate the binding activity of the VHH-Fc candidate antibody to monkey CTLA-4, the FACS method was used to detect its binding activity to monkey CTLA-4 protein on monkey CTLA-4-CHO cells. The specific method is as follows:

[0394] 1) Collect cultured monkey CTLA-4-CHO cells, centrifuge at 300g to remove the culture medium, resuspend the cells in prepared FACS buffer, count the cells, and adjust the cell suspension density to 2×10⁻⁶. 6 cells / mL;

[0395] 2) Add 100 μL of monkey CTLA-4-CHO cells to each well of a 96-well round-bottom plate, centrifuge at 300g to remove the supernatant;

[0396] 3) Add serially diluted candidate antibody and control antibody dilution to the corresponding wells, blow the cells evenly with a pipette and incubate at 4°C for 30 minutes;

[0397] 4) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200μL to the corresponding well and resuspend the cells using a multi-channel pipette;

[0398] 5) Repeat step 4 twice, centrifuge at 300g and discard the supernatant;

[0399] 6) Add 100 μL of CTLA-4 protein dilution buffer (200 nM) to the corresponding well, resuspend the cells and incubate them at 4°C for 30 minutes;

[0400] 7) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200μL to the corresponding well and resuspend the cells using a multipipe.

[0401] 8) Repeat step 7) twice, centrifuge at 300g and discard the supernatant;

[0402] 9) Add PE-labeled anti-human Fc flow cytometry antibody (Abcam, 98596), mix the cells with a pipette and incubate at 4°C for 30 minutes;

[0403] 10) Centrifuge at 300g to remove supernatant, add FACS buffer and resuspend cells;

[0404] 11) Repeat step 10) twice, add 200 μL of FACS buffer to each well, resuspend the cells, and detect them by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0405] like Figure 5A and Figure 5B As shown in the FACS experiment, the VHH-Fc chimeric antibodies obtained by fusing the eight VHH molecules screened in Example 3 with Fc all showed good activity in recognizing monkey CTLA-4 and exhibited cross-reactivity with monkey CTLA-4.

[0406] Example 8. Validation of the in vitro biological activity of chimeric VHH-Fc antibody

[0407] Staphylococcal enterotoxin B (SEB), as a superantigen, can activate T cells under extremely low concentrations, leading to a high expression of CTLA-4 protein on the T cell surface. B lymphocytes, macrophages, peripheral blood monocytes (PBMCs), and dendritic cells in peripheral blood microorganisms (PBMCs) express CD80 or CD86 proteins on their surface, which bind to CTLA-4 protein on the surface of activated T cells, thereby inhibiting T cell activation. Based on this principle, a chimeric VHH-Fc antibody and SEB were added to PBMC cells. The chimeric VHH-Fc antibody blocks the binding of CTLA-4 to CD80 or CD86, thus maintaining the activated state of T cells. The activation ability of the chimeric VHH-Fc antibody in stimulating PBMC cells in vitro was assessed by detecting the levels of corresponding cytokines (IL-2, TNF-α), as follows:

[0408] 1) PBMCs were obtained through in vitro sorting using Ficoll, and then the cell suspension density was adjusted to 2.5 × 10⁻⁶ cells / mL in 1640 RPMI medium supplemented with 10% FBS, 1% penicillin / streptomycin, 1% non-essential amino acids (NEAA), and 1% sodium pyruvate. 6 cells / mL;

[0409] 2) Add PBMC cells at a rate of 100 μL per well to a 96-well cell culture plate;

[0410] 3) Add CTLA-4 antibody dilution buffer and 10 ng / mL SEB (superantigen) mixture to the corresponding wells, and then incubate at 37°C and 5% CO2 for 72 hours;

[0411] 4) Collect cell supernatant and use an ELISA kit to detect the secretion levels of IL-2 and TNF-α in the supernatant;

[0412] The results of IL-2 secretion testing are shown in Table 9A below. Figure 6A and Table 9B and Figure 6B As shown. Compared with the negative control, the eight antibodies tested were able to activate the immune response of PBMC cells, causing them to secrete IL-2. From Figure 6A and Figure 6B It is evident that when SEB is 10 ng / mL, the levels of IL-2 produced by PBMCs stimulated by the eight candidate antibodies all exceeded or were comparable to those of the positive antibody.

[0413] Table 9A IL-2 secretion levels

[0414]

[0415] Table 9B IL-2 secretion levels

[0416]

[0417] The results of TNF-α secretion detection are shown in Table 10A below. Figure 7A and Table 10B and Figure 7B As shown. Compared with the negative control, the eight antibodies tested were able to activate the immune response of PBMC cells, causing them to secrete TNF-α. From Figure 7A and Figure 7B It is evident that when SEB concentration was 10 ng / mL, the levels of TNF-α produced by PBMCs stimulated by the eight candidate antibodies all exceeded or were comparable to those of the positive control antibody.

[0418] Table 10A TNF-α secretion levels

[0419]

[0420] Table 10B TNF-α secretion levels

[0421]

[0422] Example 9. Humanization of Antibodies

[0423] The antibodies NB25B-17 and NB25gb-1 were selected for humanization. The specific modification sites are shown in the sequence listing.

[0424] Biological activity was detected in NB25B-17-Fc and its three humanized antibodies NB25B-17-H1-IgG1, NB25B-17-H2-IgG1, and NB25B-17-H3-IgG1; and in NB25gb-1-Fc and its three humanized antibodies NB25gb-1-H1-IgG1, NB25gb-1-H2-IgG1, and NB25gb-1-H3-IgG1. The methods used for biological activity testing were consistent with those used in Examples 5, 6, 7, and 8.

[0425] Figure 8 This demonstrates the binding affinity of the humanized antibody molecule to human CTLA-4 expressed on cells. Figure 9 The study demonstrated the blocking activity of the humanized antibody molecule against the binding of CTLA-4 to CD80. Figure 10 The study demonstrated the blocking activity of the humanized antibody molecule against the binding of CTLA-4 to CD86. Figure 11 The binding affinity of the humanized antibody molecule to monkey CTLA-4 expressed on cells was demonstrated.

[0426] The antitumor activity of humanized antibodies was demonstrated in vivo using NB25B-17-H1-IgG1 and NB25gb-1-H1-IgG1 antibodies in animal models. Specifically, a tumor animal model was established by inoculating mice with human breast cancer cells MDA-MB-231 (ATCC). Mice were divided into groups and administered either NB25B-17-H1-IgG1 antibody or NB25gb-1-H1-IgG1 antibody or purified human IgG1 isotype control recombinant antibody (Biolegend, catalog number 403502), and the tumor volume of the tumor-bearing animals was measured periodically.

[0427] The results showed that NB25B-17-H1-IgG1 antibody and NB25gb-1-H1-IgG1 antibody could significantly reduce the tumor volume in tumor-bearing mice.

[0428] Although certain representative embodiments and details have been shown for illustrative purposes, it will be apparent to those skilled in the art that various changes and modifications can be made to them without departing from the scope of the subject matter. In this respect, the scope of the invention is defined only by the following claims.

[0429] sequence list

[0430] Table A lists the names and sequences of single-domain antibodies (the boxes contain the CDR sequences determined by the AbM number).

[0431]

[0432]

[0433]

[0434] Table B lists the names of single-domain antibodies and their CDR sequences.

[0435]

[0436]

[0437] Table C lists the names of humanized antibodies and their CDR sequences.

[0438]

[0439] Table D lists the names of single-domain antibodies and their FR sequences.

[0440]

[0441]

Claims

1. A CTLA-4 binding molecule comprising at least one single-domain antibody (sdAb) moiety specifically binding to CTLA-4, wherein the sdAb moiety comprises The amino acid sequences SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3); or The amino acid sequences SEQ ID NO: 15 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3); or The amino acid sequences shown in SEQ ID NO: 15 are CDR1, SEQ ID NO: 17 are CDR2, and SEQ ID NO: 6 are CDR3.

2. The CTLA-4 binding molecule of claim 1, wherein the sdAb portion comprises (i) The amino acid sequence shown in SEQ ID NO:22; or (ii) An amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

22.

3. The CTLA-4 binding molecule according to claim 2, wherein the sdAb portion comprises the amino acid sequence of SEQ ID NO: 31, 32 or 33.

4. The CTLA-4 binding molecule according to any one of claims 1 to 3, wherein the sdAb portion is linked to the Fc region of an immunoglobulin at the N-terminus or C-terminus.

5. The CTLA-4 binding molecule according to claim 4, wherein the sdAb portion is linked to the Fc region of IgG at the N-terminus or C-terminus.

6. The CTLA-4 binding molecule of claim 5, wherein the sdAb portion is linked at the N-terminus or C-terminus to the Fc region of IgG1, IgG2, IgG3 or IgG4.

7. The CTLA-4 binding molecule according to any one of claims 1 to 3, having one or more of the following properties: (1) CTLA-4 is bound with high affinity, wherein the Kd of the binding between the CTLA-4 binding molecule and CTLA-4 is 10. -5 M to 10 -12 M; (2) Block the binding of CTLA-4 to CD80 and / or CD86; (3) Induces the secretion of IL-2 and / or TNF-α; (4) Keep T cells in an activated state.

8. The CTLA-4 binding molecule according to any one of claims 1 to 3, wherein it is a bispecific or multispecific antibody.

9. The CTLA-4 binding molecule according to claim 8, wherein the bispecific antibody molecule binds to CTLA-4 and PD-1 or PD-L1.

10. An isolated nucleic acid encoding the CTLA-4 binding molecule of any one of claims 1 to 9.

11. A vector comprising the nucleic acid of claim 10.

12. The carrier according to claim 11, wherein it is an expression carrier.

13. The carrier according to claim 12, wherein it is a pHEN1 carrier.

14. A host cell comprising the nucleic acid of claim 10 or the vector of any one of claims 11 to 13.

15. The host cell of claim 14, wherein the host cell is prokaryotic or eukaryotic.

16. The host cell of claim 15, wherein the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

17. The host cell of claim 16, wherein the host cell is a 293 cell or a CHO cell.

18. A method for preparing a CTLA-4 binding molecule of any one of claims 1 to 9, the method comprising culturing a host cell of any one of claims 14 to 17 under conditions suitable for expressing a nucleic acid encoding a CTLA-4 binding molecule of any one of claims 1 to 9, optionally isolating the CTLA-4 binding molecule, and optionally further comprising recovering the CTLA-4 binding molecule from the host cell.

19. A pharmaceutical composition comprising the CTLA-4 binding molecule of any one of claims 1 to 9, and optionally a pharmaceutical excipient.

20. A pharmaceutical composition comprising the CTLA-4 binding molecule of any one of claims 1 to 9, and other therapeutic agents, and optionally pharmaceutical excipients; wherein the other therapeutic agents are selected from chemotherapeutic agents, anti-PD-1 antibodies, anti-PD-L1 antibodies, or cytotoxic agents.

21. A combination product comprising the CTLA-4 binding molecule of any one of claims 1 to 9, and one or more other therapeutic agents, wherein the other therapeutic agents are selected from chemotherapeutic agents, anti-PD-1 antibodies, anti-PD-L1 antibodies, or cytotoxic agents.

22. Use of the CTLA-4 binding molecule of any one of claims 1 to 9, the pharmaceutical composition of claim 19 or 20, or the combination product of claim 21, for the preparation of a medicament for the prevention or treatment of breast cancer in a subject.

23. A kit for detecting CTLA-4 in a sample, said kit comprising the CTLA-4 binding molecule of any one of claims 1 to 9, for performing the following steps: (a) Contacting the sample with the CTLA-4 binding molecule of any one of claims 1 to 9; and (b) Detect the formation of the complex between the CTLA-4 binding molecule and CTLA-4; optionally, the CTLA-4 binding molecule is detectably labeled.

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