TW202016145A - Humanized antibodies against human tim-3 and uses thereof - Google Patents

Abstract

A humanized anti-human T-cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody, or a binding fragment thereof, wherein the antibody or the binding fragment includes a heavy-chain variable domain that comprises framework region sequences derived from a human immunoglobulin and the following complementarity determining region (CDR) sequences: HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4), HCDR3 (SEQ ID NO: 5), and a light-chain variable domain that includes framework region sequences from a human immunoglobulin and the following CDR sequences: LCDR1 (SEQ ID NO: 6), LCDR2 (SEQ ID NO: 7), and LCDR3 (SEQ ID NO: 8).

Description

Humanized antibodies against human TIM-3 and their uses

The present invention relates to methods for producing humanized antibodies that specifically bind to human T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and the use of these antibodies.

The immune response plays an important role in resisting cancer. Therefore, T cell failure may be related to tumor growth in the host. T cell failure is caused by multiple mechanisms. Planned cell death molecule (PD-1) is a marker of depleted T cells. Blocking the interaction between PD-1 and its ligand (PD-1 ligand, or PD1L, or PD-L1) can partially restore T cell function.

In addition to PD-1, T-cell immunoglobulin mucin 3 (TIM-3) was found on CD8 ⁺ tumor-infiltrating lymphocytes in mice with solid tumors. TIM-3 was originally thought to be a T helper cell (Th) 1-specific type I membrane protein. TIM-3 (an immune checkpoint) regulates macrophage activation and plays an important role in Th1 immunity and tolerance induction.

It was also found that all tumor infiltrating lymphocytes expressing TIM-3 express PD-1. These lymphocytes expressing TIM-3 and PD-1 account for the majority of T cells infiltrating the tumor. In addition, these lymphocytes expressing TIM-3 and PD-1 also exhibit the most severe failure phenotype: they cannot proliferate and cannot produce IL-2, TNF, and IFN-γ. (Sakuishi et al., J. Exp. Med., 2010, 207(10): 2187-2194).

Recently, TIM-3 has also been found to regulate other cells, such as Th17 cells, CD4(+) CD25(+) regulatory T cells (T _reg ), CD8(+) T cells, and certain innate immune cells.

Because the TIM-3 pathway is involved in the pathogenesis of autoimmune diseases, chronic viral infections, and cancer, it is necessary to find agents that can inhibit or block the TIM-3 signaling pathway.

The embodiments of the present invention relate to humanized antibodies that can specifically bind to TIM-3 and thereby inhibit the function of TIM-3. It was found that TIM-3 signaling pathway is related to T cell failure. Therefore, the antibodies of the present invention can be used to prevent or treat diseases or conditions associated with T cell failure, such as autoimmune diseases and cancer.

One aspect of the present invention relates to humanized antibodies against human T cell immunoglobulin domain and mucin domain 3 (TIM-3) or binding fragments thereof (eg, scFv, Fab, or (Fab) ₂ ). According to an embodiment of the present invention, the humanized anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof may comprise a heavy chain variable domain and a light chain variable domain , The heavy chain variable domain comprises the framework region sequence derived from human immunoglobulin and the following complementarity determining region (CDR) sequences: HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4), HCDR3 (SEQ ID NO: 5), and the light chain variable domain comprises the framework sequence from human immunoglobulin and the following CDR sequences: LCDR1 (SEQ ID NO: 6), LCDR2 (SEQ ID NO: 7) and LCDR3 (SEQ ID NO: 8). The framework region sequence in the heavy chain variable domain can be derived from the corresponding framework region sequence of IGHV1-2*02, and the framework region sequence in the light chain variable domain can be derived from the corresponding framework region sequence of IGVK4-1*01 .

According to an embodiment of the present invention, the anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof may comprise a heavy chain having the sequence of SEQ ID NO: 10 or 20 or 21 The variable domain and the light chain variable domain having the sequence of SEQ ID NO: 9 or 22.

According to the embodiments of the present invention, the antibodies or binding fragments of the present invention can be used for the diagnosis, prognosis and treatment of cancers expressing TIM-3 on the cell surface. Such cancers include, for example, lung cancer, liver cancer, esophageal cancer, and solid tumors.

Other aspects and advantages of the present invention will be apparent from the following description and the scope of the attached patent application.

definition

As used herein, "complementarity determining region (CDR)" refers to the three hypervariable sequences in the variable region that are involved in antigen recognition. The three CDRs are dispersed in the light or heavy chain variable regions using four "framework" regions. The CDR is mainly responsible for binding to the epitope of the antigen. The CDRs of each chain are usually called (continuous numbering from the N-terminus) CDR1, CDR2, and CDR3, and are usually identified by the chain in which the specific CDR is located. Thus, V _H CDR3 is located in the heavy chain variable domain of the antibody in which it was found, and V _L CDR1 is CDR1 from the light chain variable domain of the antibody in which it was found. These may be referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, where H indicates a heavy chain and L indicates a light chain.

The sequences of the framework regions (FR) of different light or heavy chains are relatively conserved within the species. The framework region of the antibody (that is, the combined framework region of the composed light chain and heavy chain) is used as a framework for positioning and aligning the CDR in three-dimensional space. The four FRs flanked by the three CDRs can be called FR1, FR2, FR3, and FR4. The antibodies of the present invention may include framework region sequences "derived from" human immunoglobulins. As used herein, the term "derived from" includes framework sequences that are identical or at least 90% identical to the sequences of the original framework sequences of human antibodies. In some embodiments, the antibodies of the invention may comprise framework region sequences "selected" from human immunoglobulins. As used herein, the term "selected" includes framework sequences that are identical to the sequences of the original framework sequences of human antibodies.

The amino acid sequence of the CDR and framework region (FR) can be determined using various well-known definitions in the art, such as Kabat, International ImmunoGenetics Database (IMGT). The CDR regions described herein are based on Kabat definitions.

The embodiments of the present invention relate to humanized antibodies that specifically bind to human TIM-3. These humanized anti-human TIM-3 antibodies can be used to treat and/or diagnose immune diseases or cancerous diseases.

The immune response plays an important role in resisting cancer. However, in chronic viral infections and cancer, T cell failure has been found to be associated with these diseases. T cell failure is caused by multiple mechanisms. Planned cell death molecule (PD-1) is a marker of depleted T cells. Blocking the interaction between PD-1 and its ligand (PD-1 ligand, or PD1L, or PD-L1) can partially restore T cell function, indicating that blocking PD-1 and PD-L1 interaction may be An effective method of restoring immune function, thereby treating or preventing diseases associated with PD-1 and/or PD-L1 mediated immunosuppression or failure.

In addition to PD-1, T cell immunoglobulin mucin 3 (TIM-3), a marker of immune checkpoints, was also found to be associated with T cell failure. TIM-3 was originally thought to be a T helper cell (Th) 1-specific type I membrane protein. TIM-3 (an immune checkpoint) regulates macrophage activation and plays an important role in Th1 immunity and tolerance induction.

TIM-3 was also found on CD8 ⁺ tumor infiltrating lymphocytes in mice with solid tumors. It was also found that all tumor infiltrating lymphocytes expressing TIM-3 express PD-1. These lymphocytes expressing TIM-3 and PD-1 account for the majority of T cells infiltrating the tumor. In addition, these lymphocytes expressing TIM-3 and PD-1 also exhibit the most severe failure phenotype: they cannot proliferate and cannot produce IL-2, TNF, and IFN-γ. (Sakuishi et al., J. Exp. Med., 2010, 207(10): 2187-2194). In dysfunctional CD8 ⁺ T cells and Tregs in cancer, the TIM-3 pathway may interact with the PD-1 pathway. After PD-1 inhibition, TIM-3 mainly appears on activated CD8 ⁺ T cells and inhibits macrophage activation. After anti-PD-1 therapy, upregulation of TIM-3 will be observed in progressive tumors. (Koyama S. et al. (February 2016), "Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints" Nature Communications, 7 : 10501, doi: 10.1038/ncomms10501). This phenomenon seems to be a form of strain resistance to immunotherapy.

Recently, TIM-3 has been found to regulate other cells, such as Th17 cells, CD4(+) CD25(+) regulatory T cells (T _reg ), CD8(+) T cells, and certain innate immune cells. Because the TIM-3 pathway is involved in the pathogenesis of autoimmune diseases, chronic viral infections, and cancer, the antibodies of the present invention can be used to treat these diseases.

According to embodiments of the invention, there are several pure lines that produce antibodies against human TIM-3. Briefly, BALB/c mice were presensitized with purified recombinant human TIM-3 antigen (as a fusion protein with a 6×His tag). Spleen cells are harvested and then fused with Fo cells and then cultured. Fusion tumor cells that can secrete monoclonal antibodies that recognize TIM-3 antigen are selected by TIM-3 antigen-based ELISA. The selected pure lines were verified by FACS analysis. Isolate and analyze many pure lines. Determine the variable domain sequence.

Further study of an exemplary antibody 8C11. The 8C11 epitope was found to bind to the extracellular domain of TIM-3 in the region of RKGDVSL (SEQ ID NO: 11) and/or EKFNLKL (SEQ ID NO: 12). It was found that 8C11 can bind to TIM-3 and interfere with the interaction between TIM-3 and its ligand galectin-9. In addition, 8C11 binding to TIM-3 does not induce T cell death (apoptosis), that is, antibody binding to TIM-3 does not trigger the TIM-3 signaling pathway. Therefore, there is no risk of T cell failure induced by the antibody of the present invention. This is important because in order to be used as a therapeutic agent, antibodies that block the interaction between TIM-3 and its ligand (galectin-9) should not activate the TIM-3 signaling pathway itself, resulting in T cell depletion /Apoptosis.

In addition, the antibody of the present invention can promote the secretion of IFN-γ and TNF-α by T cells, thereby enhancing the immune response. These antibodies can inhibit tumor growth in animal models. Therefore, the antibody of the present invention can be used to treat cancer, such as lung cancer, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, or prostate cancer.

Although the above anti-system is useful, it is derived from mice. When used in humans, these mouse antibodies may cause complications (eg, adverse immune reactions). In addition, these mouse antibodies do not induce antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) responses, which helps eliminate cancer cells or infected cells. Therefore, in order to further develop these antibodies for clinical use, these antibodies have been humanized. In addition, humanized antibodies have been optimized using back mutations and mutations in the CDR sequences.

The binding of humanized antibodies to various Tim family members and Tim-3 from different species has been analyzed. It was found that the humanized antibody of the present invention is specific to TIM-3 and does not bind to other TIM family members (for example, TIM-1 and TIM-4), and some antibodies (for example, hu8C11) are species-specific ( For example, it only binds to human TIM-3), while other variants can also bind to TIM-3 from other species (eg monkeys). In addition, the ability of humanized antibodies to inhibit tumor growth in xenograft systems has been tested. The antibody of the present invention was found to be effective in inhibiting tumor growth in vivo, and combination therapy with other chemotherapeutic agents (eg, cyclophosphamide) can provide a synergistic effect. The following will describe these contents with specific examples. Those skilled in the art will understand that these examples are for illustration only and are not meant to limit the scope of the present invention. Selection of 8C11 monoclonal antibody

The selection of the gene encoding 8C11 mAb was performed according to the method described below. Although the following procedures may state specific conditions and parameters, those skilled in the art should understand that this is only an example for illustrating embodiments of the present invention, and other modifications and variations are possible without departing from the scope of the present invention of. Cloning and preparation of cDNA for antibody genes

The fusion tumor is cultured in a medium with a defined composition. When the cell number reached about 10×10 ⁶ cells/ml, the cells were collected by centrifugation, and then a TRIzol kit was added to extract total RNA according to the instruction manual (Thermo Fisher Scientific, Waltham, MA, USA). The variable region of antibody cDNA was selected using mouse Ig-primer set according to the supplier's instruction manual. Using 5 micrograms of total RNA as a template, 50 ng/ml random primers, and 10 µM dNTPs, it was mixed with diethyl pyrocarbonate (DEPC)-treated water in a PCR tube to prepare the first strand of cDNA.

The reaction mixture was incubated at 65°C for 5 minutes, and then placed on ice. Gently mix 10 (ten) µl cDNA synthesis mixture containing 2 µl 10× room temperature buffer, 4 µl 25 mM MgCl ₂ , 2 µl DTT, 1 µl 4 units RNaseOUT (Thermo Fisher Scientific) and 1 µl 200 units SuperScript III RT and collected by brief centrifugation. The reaction tube was incubated at 25°C for 10 minutes, followed by 50°C for 50 minutes. The reaction was terminated by heating at 85°C for 5 minutes and then the tube was cooled on ice. The tube was briefly centrifuged to collect the reaction product, and 1 µl of RNase H was added, and the mixture was incubated at 37°C for 20 minutes.

A reaction mixture consisting of 5 µl cDNA, 5 µl 10× reaction buffer, 1 µl 10 mM dNTP mixture, 1 µl 2.5 units Taq polymerase and 1 µl forward and reverse primers was prepared with double distilled water to a final 50 µl Volume and undergo PCR. For the amplification of antibody light and heavy chains, 94°C is the first step, and then the cycle of maintaining 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute is repeated 30 times. After the reaction cycle, the final step was 72°C for 10 minutes. The reaction mixture was analyzed using 2% agarose gel. The product with the predicted molecular weight was conjugated to the selection vector and then used to determine the nucleotide sequence.

Based on the sequence information, the ExPASY Translation Tool (ExPASY Bioinformatics Resource Portal) is used to translate the antibody sequence into the protein sequence. The sequence of the obtained anti-human TIM-3 antibody 8C11 includes a heavy chain amino acid sequence and a light chain sequence. The complementarity determining regions (CDRs) in these sequences were determined using Kabat et al., "Sequences of proteins of immunological interest", Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Volumes 1-3 .

As shown in FIG. 1, the amino acid sequence of the 8C11 antibody contains a heavy chain variable region (SEQ ID NO: 1) and a light chain variable region (SEQ ID NO: 2). Framework regions (FR1, FR2, FR3 and FR4) and CDR (HCDR1 SEQ ID NO: 3, HCDR2 SEQ ID NO: 4, HCDR3 SEQ ID NO: 5, LCDR1 SEQ ID NO: 6, LCDR2 SEQ ID NO: 7 and LCDR3 SEQ ID NO: 8) as indicated. Humanization of anti-TIM3 antibody 8C11 mAb

8C11 VL(CDR 序列顯示在方框中： LCDR1 、 LCDR2 及 LCDR3) ：

To make anti-TIM3 antibodies useful in the clinic, 8C11 was chosen for humanization. Humanization begins by selecting human antibody sequences that are highly homologous to the 8C11 sequence. The homology search can use the complete sequence of 8C11, only the variable domain sequence of 8C11, or only the framework sequence of 8C11. For example, the VH and VL sequences of mAb 8C11 shown below can be used to search the IMGT database for human antibody sequences with the highest homology. 8C11 VH (CDR sequence is shown in the box: HCDR1 , HCDR2 and HCDR3) :

8C11 VL (CDR sequence is shown in the box: LCDR1, LCDR2 and LCDR3):

Alternatively, only the 8C11 framework sequence (ie, no CDR sequence) can be used in the homology search of the IMGT database.

Based on these searches, IGHV1-2*02 (IMGT deposit number X62106) and IGVK4-1*01 (IMGT deposit number Z00023) were selected as the most homologous human sequences of the heavy chain variable domain and the light chain variable domain, respectively . Next, the humanization of 8C11 is based on the framework sequences from these two sequences.

Next, the CDR sequence from 8C11 was used to replace the corresponding CDR sequence in the human antibody sequence. The CDR regions in the human sequence can be obtained from the analysis using methods known in the art, such as the Kabat method. PCR or similar techniques can be used to graft mouse CDR sequences into human antibody sequences.

Figure 2 shows the full-length light chain (VL+VC; SEQ ID NO: 13) and full-length heavy chain (VH+CH1+CH2+CH3; SEQ ID NO: 14) of the humanized antibody Hu8C11, in which the light chain variable domain ( VL) sequence (SEQ ID NO: 9) and heavy chain variable domain (VH) sequence (SEQ ID NO: 10) are shown in bold.

Next, the humanized anti-Tim3 antibody (that is, Hu8C11) was subcloned into the antibody expression vector. Any suitable vector known in the art may be used, such as the TGEX vector from Antibody Design Labs (San Diego, CA). The expression vector is then used to transfect cells, such as FreeStyle 293-F cells (Thermo Fisher Scientific), to express antibodies. 3) Revert mutation to improve binding.

Transplantation of mouse CDR sequences onto human frameworks/antibodies produces variable domains (VH and VL) containing sequences from two different sources (ie, mouse CDR sequences + human framework sequences). Such heterologous domains may not have optimal sequences, and may have structural disturbances that may affect antibody binding. Therefore, the affinity of the antibody may not be the best. To improve binding affinity, some amino acids in the variable domain can be mutated back to mouse residues.

In one method of selecting amino acids for back-mutation, computer model creation can be used to analyze key amino acid residues that may affect antibody binding. In the establishment of the model, it is also possible to apply prior knowledge derived from antibody engineering based on successful cases. Based on model establishment, selective amino acid substitutions can be made, for example, replacing the amino acid residues in Hu8C11 with the corresponding amino acid residues in the original murine 8C11 (ie, back mutation). Next, mutant antibodies can be screened, expressed and evaluated for binding to select improved antibodies.

3A to 3E show the nucleotide and protein sequences of the heavy and light chain regions of Hu8C11 and several back mutation mutants of Hu8C11. Figure 3A shows the Hu8C11-H sequence of the heavy chain variable region sequence of the initial humanized mAb Hu8C11 (nucleotide: SEQ ID NO: 15; protein: 10). Figure 3D shows the Hu8C11-L sequence (nucleotide: SEQ ID NO: 18; protein: 9) of the light chain region sequence of the initial humanized mAb Hu8C11. Figures 3B and 3C show that two improved heavy chain variants (Hu8C11-HB1; nucleotide: SEQ ID NO: 16; protein: SEQ ID NO: 20) and (Hu8C11-HB2; nucleotide: SEQ ID NO: 17; protein: SEQ ID NO: 21), and an improved light chain variant (Hu8C11-LB3; nucleotide: SEQ ID NO: 19; protein: SEQ ID NO: 22).

The Hu8C11-HB1 (SEQ ID NO: 20) variant contains back mutations at amino acid residues 38, 48, 66, 67, 71, 73, and 76 (replacing the original residue in Hu8C11 with the murine 8C11 mAb Corresponding residue). The Hu8C11-HB2 (SEQ ID NO: 21) variant contains back mutations at amino acid residues 69, 71, 73 and 76. The Hu8C11-LB3 (SEQ ID NO: 22) variant contains back mutations at amino acid residues 46 and 49.

Antibodies containing different combinations of variable heavy and light chains were produced in 293 cells. These antibodies were purified and tested for binding to human Tim3. The combination includes Hu8C11-B1B3 (containing Hu8C11-HB1 heavy chain and Hu8C11-LB3 light chain), Hu8C11-B2B3 (containing Hu8C11-HB2 heavy chain and Hu8C11-LB3 light chain) and Hu8C11-HB3 (containing original Hu8C11 heavy chain and Hu8C11- LB3 light chain). Binding analysis can be performed using any method known in the art, such as ELISA, BIAcore or FACS analysis. For example, Table 1 shows the BIAcore analysis results of these combination variants as well as the original murine mAb (8C11-MM) and the initial humanized mAb (Hu8C11-HH). Table 1

As shown in Table 1, the binding affinity of the initial humanized mAb (Hu8C11-HH) is slightly lower than that of the original murine mAb (8C11-MM). Various combinations of back-mutated variants improved slightly in terms of binding affinity. For example, the Hu8C11-B1B3 mutant has substantially the same affinity as the murine mAb (8C11-MM). The fact that these back-mutated variants retain binding affinity indicates that the humanized anti-human TIM-3 antibody can adapt to some degree of mutation in the framework sequence. Therefore, the antibodies of the present invention may contain framework region sequences "derived from" human immunoglobulins. As used herein, the term "derived from" includes framework sequences that are identical or at least 90% identical to the sequences of the original framework sequences of human antibodies.

In order to test the specificity of humanized antibodies, we tested the binding of Hu8C11 to different Tim molecules (ie, Tim1, Tim3, and Tim4). The function of different Tim molecules in immune regulation is complex, and different Tim molecules may have different roles. Therefore, in order to use anti-Tim3 antibodies to reverse T cell failure, it is expected that the antibodies are specific for Tim3 and will not act on Tim1 or Tim4. As shown in FIG. 4, Hu8C11 and control antibodies (anti-Tim3 antibody, pure line 2E2, which are available from commercial sources such as Millipore Sigma) only specifically bind Tim3 and do not bind Tim1 or Tim4. This result indicates that Hu8C11 or its variants can be safely used to reverse T cell failure by interfering with the interaction between Tim3 and its ligand (galectin-9).

In addition to BIAcore binding analysis, antibodies were also analyzed using FACS analysis. Briefly, human T cells are first activated with anti-CD3 and anti-CD28 coated magnetic beads, which are commercially available (such as Dynabeads® human T-activating factor CD3/CD28 from Thermo Fisher Scientific). Anti-CD3 and anti-CD28 antibodies provide primary and costimulatory signals and are optimized for effective T cell activation and expansion.

T cell activation using anti-CD3/anti-CD28 magnetic beads was performed according to the procedures of commercial providers. For example, using Dynabeads® human T-activating factor CD3/CD28 from Thermo Fisher Scientific, the following is an exemplary procedure:

For example, start with 8×10 ⁴ purified T cells in 100 to 200 μL of culture medium in a 96-well tissue culture dish. Add 2 μL of pre-washed and resuspended Dynabeads® to obtain a 1:1 bead to cell ratio. Incubate at 37°C in a humidified CO ₂ incubator. Activated T cells were collected and the magnetic beads were removed, followed by FACS analysis.

After activation, T cells are incubated with test antibodies, which include commercially available 2E2 antibodies as a positive control. The binding of antibodies to T cells was performed in a humidified CO ₂ incubator at 37°C for 1 hour. Next, a fluorescent-labeled secondary antibody (ie, APC-labeled anti-mouse IgG or FITC-labeled anti-human IgG) is added. The secondary antibody binding was allowed to proceed for 1 hour. Next, the cells were analyzed by FACS. As shown in Figure 5, all antibodies, namely 2E2, 8C11 and Hu8C11, bind well to T cells. This result indicates that humanized Hu8C11 retains the binding affinity for activated T cells. 4) CDR affinity optimization and alanine scanning of key amino acid residues.

In addition to the above back mutations in the framework regions, antibody affinity can be further improved by optimizing CDR sequences. First, we performed alanine scanning on the residues in the CDR to identify the residues that are critical for binding. Various alanine-substituted variants are induced by site-directed mutagenesis and are temporarily expressed in F293 cells.

The performance of these mutants can be checked for proper performance, for example using dot blots. In short, serial dilutions of Hu8C11 can be used to establish a standard curve within the expected protein production range, as shown in Figure 6A.

The following table summarizes the results of dot blot analysis of alanine mutants of CDR residues in the heavy chain (Figure 6B):

The following table summarizes the results of dot blot analysis of alanine mutants of light chain CDR residues (Figure 6C):

Various alanine mutants are constructed in expression vectors, which are temporarily expressed in suitable cells, such as 293 cells or CHO cells. Next, the binding affinity of the mutant variant can be analyzed by any suitable method, such as analysis using ELISA, Biacore or FACS. Figure 7 shows representative FACS results of heavy chain alanine mutants, indicating that the G26A mutant loses binding, while Y27A retains binding to Tim3. All alanine mutants (G26A, Y27A, T28A, F29A, T30A, D31A, Y32A, Y33A, M34A, N35A, R50A, V51A, P53A, S54A, N551, G56A, G57A, N63A, F64A, K65A, G66A, R99A, D100A, S101A, S102A, G103A, Y104A, W105A, F106A and Y108A) are all tested similarly. From these heavy chain alanine scans and FACS analysis, it was found that among the heavy chain mutants, the G26A, R99A, S102A and W105A mutants lost their binding, indicating that these residues in the heavy chain CDR bind to Tim-3. It is very important.

Similarly, Figure 8 shows representative FACS results of the light chain alanine mutant, indicating that the K24A mutant retains binding, while the S26A mutant loses binding to Tim3. All alanine mutants (K24A, S26A, Q27A, V29A, S30A, T31A, V33A, V34A, S50A, P51A, S52A, Y53A, R54A, T56A, Q89A, Q90A, H91A, Y92A, N93A, I94A, P95A, W96A and T97A) are similarly tested. From the light chain alanine scan and FACS analysis, it was found that among the light chain mutants, S26A, V29A, S30A, T31A, H91A, and N93A lost their binding to Tim3, indicating that these residues in the light chain CDR are related to Tim The combination of -3 is crucial.

As described above (FIG. 4A), mAb 8C11 and Hu8C11 are specific to Tim-3 and do not bind to Tim-1 and Tim-4. In addition, these anti-systems were found to be species-specific. To test this, a monkey TIM-3 (TIM-3 ECD-GFP) labeled with green fluorescent protein (GFP) was constructed. The expression vector used to temporarily express this recombinant protein was transfected into F293 cells. Transfected F293 cells expressing TIM-3 ECD-GFP were reacted with antibodies, including control 2E2 mAb and 8C11 mAb, and analyzed by FACS. Wild-type F293 cells and anti-mouse IgG APC-conjugate were used as negative controls. As shown in Figure 9, mAb 2E2 can bind monkey TIM-3, while 8C11 mAb cannot. These results indicate that the 8C11 mAb is species-specific; it binds human TIM-3, but not monkey TIM-3.

Various alanine mutants in the CDR regions were also tested for binding to monkey TIM-3. It was unexpectedly found that some Hu8C11 mutants containing alanine mutations in the heavy chain CDRs can bind monkey TIM-3. For example, Figure 10 shows that the VH-S101A mutant and the VH-S102A mutant of Hu8C11 can bind to monkey TIM-3. Similarly, VH-T28A and VH-W105A can also bind to monkey TIM-3. These results indicate that these CDR mutations (T28A, S101A, S102A and W105A) change the binding properties of Hu8C11.

Similar FACS analysis was performed with light chain alanine mutants. The S26A mutant in the light chain CDR was found to bind monkey TIM-3, indicating that this mutant also has altered binding properties.

O指示結合；X指示沒有結合；N指示不可用。The following table summarizes the results of FACS and dot blot analysis of various heavy chain mutants of Hu8C11:

O indicates bonding; X indicates no bonding; N indicates unavailable.

O指示結合；X指示沒有結合；N指示不可用。 異種移植動物模型說明Hu8C11之腫瘤生長抑制能力The following table summarizes the results of FACS and dot blot analysis of various light chain mutants of Hu8C11:

O indicates bonding; X indicates no bonding; N indicates unavailable. Xenotransplantation animal model demonstrates the tumor growth inhibitory ability of Hu8C11

Tumor growth is often associated with T cell failure. T cell failure causes a reduction in the immune response, which allows cancer cells to grow uncontrollably. Because anti-TIM-3 antibodies were found to reverse or minimize T cell failure, such as blocking galectin-9/TIM-3 binding and enhancing T cell IFN-γ and TNF-α Secreted, so these antibodies may prevent or slow the growth of cancer cells. To test this, the following experiment was conducted.

11A, on day 0 of the ^experiment, NOD.Cg- Prkdc ^scid Il2rg tmlwjl / YckNarl mice (5-6 weeks old) were subcutaneously (sc) injection of highly metastatic human lung cancer cells CLl-5 (total ¹⁰⁶ Cells/100 μl in PBS). The mice were randomly divided into groups and received different treatments.

As shown in FIG. 11A, 0.5×10 ⁶ human fresh PBMC were injected (ip) on days 13 and 20, and at the same time on days 13, 15, 17, 20, 23 and 23 Treatment with humanized anti-TIM-3 mAb (Hu8C11, 1 mpk, 3 mpk or 10 mg/kg per injection) was injected on 25 days. For the group receiving cyclophosphamide, cyclophosphamide was administered on day 12 (50 mg/kg per injection).

Survival and xenograft versus host response were monitored daily until 28 days. Animals with clinical symptoms of xenograft versus host disease (xGVHD) (>15% weight loss, hunchback posture, decreased mobility, hair loss, shortness of breath) were sacrificed and the end of survival was recorded. The mice were sacrificed on day 28 and the subcutaneous tumors were removed, the tumors were weighed, and processed for immunohistochemistry (IHC) and FACS analysis.

As shown in FIG. 11B, cyclophosphamide or anti-TIM3 can each inhibit tumor growth. Cyclophosphamide is a chemotherapeutic agent. The combination therapy using cyclophosphamide and anti-TIM3 antibody produced a synergistic effect and tumor growth was significantly inhibited. These results indicate that the antibodies of the present invention can be effective cancer therapeutic agents, especially when used together with another immunosuppressive agent or cancer therapeutic agent.

Some embodiments of the present invention relate to the use of the antibodies of the present invention for the treatment of diseases or disorders associated with T cell failure mediated by TIM-3. Such diseases include cancers such as lung cancer, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, or prostate cancer. According to an embodiment of the present invention, a method for treating such cancer may include administering an effective amount of the antibody of the present invention to an individual in need. The effective amount is the amount needed to achieve treatment. Those skilled in the art should understand that the effective amount will depend on the disease, patient (age, weight), dosage form, route of administration, etc. Those skilled in the art can determine the effective amount without undue experimentation. Administration can be by any suitable means, including injections, such as subcutaneous injections, intramuscular injections, intravenous injections, intraperitoneal injections, and the like.

Although the present invention has been described based on a limited number of embodiments, those skilled in the art will appreciate that other embodiments can be designed without departing from the scope of the present invention as disclosed herein. Therefore, the scope of the present invention should be limited only by the scope of the attached patent application.

Figure 1 shows the sequence of the heavy and light chain variable regions of a single anti-human TIM-3 antibody 8C11 according to an embodiment of the present invention. The complementarity determining region (CDR) sequences are shown as bold and underlined sequences. The framework sequences are scattered between the CDR sequences and flanked by the CDR sequences.

Figure 2 shows the full length of the heavy chain (SEQ ID NO: 14) and light chain (SEQ ID NO: 13) of the humanized anti-human TIM-3 antibody Hu8C11 according to one embodiment of the present invention (including signals shown as underlined) Peptide) sequence. The light chain variable domain (VL; SEQ ID NO: 9) and the heavy chain variable domain (VH; SEQ ID NO: 10) are shown in bold in their respective sequences, and the complementarity in the variable domain is determined Region (CDR) sequences (identical to those shown in Figure 1) are shown in boxes.

FIG. 3A shows the nucleotide and protein sequences of the heavy chain variable domain of Hu8C11 (nucleotide: SEQ ID NO: 15; protein: SEQ ID NO: 10), and FIG. 3D shows the light chain variable domain of Hu8C11 The nucleotide and protein sequences (nucleotides: SEQ ID NO: 18; protein: SEQ ID NO: 9). Also shown are three back-mutated mutant sequences: Figure 3B shows Hu8C11-HB1 (nucleotide: SEQ ID NO: 16; protein: SEQ ID NO: 20), and Figure 3C shows Hu8C11-HB2 (nucleotide: SEQ ID NO: 17; protein: SEQ ID NO: 21), and FIG. 3E shows Hu8C11-LB3 (nucleotide: SEQ ID NO: 19; protein: SEQ ID NO: 22).

Figure 4 shows that 8C11 mAb and control mAb 2E2 are specific for the Tim3 antigen line and do not bind Tim1 or Tim4.

Figure 5 shows the results of FACS analysis using activated T cells. The results showed that the mouse mAb 8C11, the humanized mAb Hu8C11 and the control mAb 2E2 were tightly bound to activated T cells.

Figure 6A shows the standard curve for dot blot analysis. Figure 6B shows the dot blot analysis of the heavy chain alanine mutant, and Figure 6C shows the dot blot analysis of the light chain alanine mutant.

Figure 7 shows representative results of FACS analysis of heavy chain alanine mutants that bind to human Tim3. These results and similar results of other alanine mutants indicate that the binding of G26A, R99A, S102A and W105A mutants is significantly impaired, indicating that these residues are essential for binding.

Figure 8 shows representative results of FACS analysis of a light chain alanine mutant that binds to human Tim3. These results and similar results of other alanine mutants indicate that the binding of S26A, V29A, S30A, T31A, H91A, N93A and W96A mutants is significantly impaired, indicating that these residues are essential for binding.

Figure 9 shows that 8C11 mAb cannot bind to monkey TIM-3 extracellular domain (ECD), while control antibody 2E2 can bind. These results indicate that 8C11 mAb is specific for human TIM-3 but not monkey TIM-3.

Figure 10 shows that the VH-S101A and VH-S102A mutants of Hu8C11 in the heavy chain CDR change the properties of the antibody so that the mutant antibody can bind to monkey TIM-3.

Figure 11A shows the treatment schedule for testing the role of Hu8C11 in a xenograft lung cancer mouse model.

FIG. 11B shows the effect of inhibiting the growth of cancer cells using a xenograft model using Hu8C11, cyclophosphamide, and a combination therapy using Hu8C11 and cyclophosphamide. The results show that the combination therapy has a synergistic effect.

Claims (10)

A humanized anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof, wherein the antibody or binding fragment comprises a heavy chain variable domain and a light chain variable domain , The heavy chain variable domain comprises the framework region sequence derived from human immunoglobulin and the following complementarity determining region (CDR) sequences: HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4), HCDR3 (SEQ ID NO: 5), and the light chain variable domain comprises the framework sequence from human immunoglobulin and the following CDR sequences: LCDR1 (SEQ ID NO: 6), LCDR2 (SEQ ID NO: 7) and LCDR3 (SEQ ID NO: 8). The anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof according to claim 1, wherein the framework region sequences in the heavy chain variable domain are derived from IGHV1- Corresponding framework sequence of 2*02. The anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof according to claim 1, wherein the framework region sequences in the light chain variable domain are derived from IGVK4- Corresponding framework sequence of 1*01. The anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises SEQ ID NO: 10 or SEQ ID NO: 20 or the sequence of SEQ ID NO: 21. The anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof according to claim 1, wherein the light chain variable domain comprises SEQ ID NO: 9 or SEQ ID NO: The sequence of 22. The anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody or binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises SEQ ID NO: 10 or SEQ ID NO: 20 or the sequence of SEQ ID NO: 21, and the light chain variable domain comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 22. A pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises the anti-human T cell immunoglobulin domain and mucin domain 3 (TIM-3) antibody according to any one of claims 1 to 6, or Combine fragments. As in the pharmaceutical composition of claim 7, the pharmaceutical composition further comprises a chemotherapeutic agent. As in the pharmaceutical composition of claim 8, the chemotherapeutic agent is cyclophosphamide. The pharmaceutical composition according to any one of claims 7 to 9, wherein the cancer is lung cancer, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, or prostate cancer.

Images