Humanized monoclonal antibodies for the restoration of human T and B cells dysfunctional in response to cancer and viral infections

Abstract

The present invention relates to two fully humanized Δ42PD1-specific mAbs, specifically huCH101 and huCH34, with a humanization rate of 97.0% by CDR grafting. The subject invention further relates to methods of using huCH101 and / or huCH34 to treat a subject in need of a Δ42PD1-specific mAb, including a subject with cancer and / or an infectious disease.

Description

[Background technology]

[0001] Programmed death-1 (PD-1, CD279) is a member of the CD28 superfamily that negatively regulates T cell function through interactions with its two native ligands, PD-L1 (CD274) and PD-L2 (CD273). PD-1 is a type I transmembrane receptor protein composed of a single immunoglobulin (Ig) variable-like domain, a cytoplasmic domain, and two tyrosine-based signaling motifs. PD-1 is constitutively expressed at low levels on resting T cells and is upregulated on T cells, natural killer T (NKT) cells, B cells, and macrophages upon activation.

[0002] Deficiency of PD-1 in several mouse models confers significant resistance to bacterial infections via innate immunity, indicating an important regulatory role of PD-1 against pathogen infections. Furthermore, PD-1 plays an important role in many autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis.

[0003] Immune checkpoint inhibitors, such as humanized programmed cell death 1 (PD-1)-specific mAbs, have been widely used in cancer immunotherapy. In addition to PD-1, alternatively spliced ​​isoforms of PD-1, namely, Δ42PD1, have been discovered. Furthermore, Δ42PD1-specific mAbs, CH101 and CH34, have been generated using hybridoma technology. The immunomodulatory effects of Δ42PD1 have also been demonstrated, suggesting that Δ42PD1 may be a target for the development of therapeutic antibodies to treat human diseases. Many cancer patients do not respond to anti-PD1 immunotherapy.

[0004] Thus, there is a need for effective anti-PD1 immunotherapies, particularly for human use. Summary of the Invention

[0005] The subject invention relates to an antibody that specifically binds to PD-1 protein isoform Δ42PD1. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the subject invention is a CH101 antibody and a CH34 antibody that are fully humanized by CDR grafting to about 97.0% humanity, i.e., huCH101 antibody and huCH34 antibody. The specificity, binding affinity, and sensitivity of the interaction with Δ42PD1 were also measured.

[0006] Another aspect of the present invention provides a method for preventing, treating or ameliorating cancer and viral infection.The method comprises administering to a subject who needs such prevention and treatment an effective amount of an antibody that targets the PD1 protein isoform of the present invention (such as Δ42PD1 protein), a nucleic acid molecule (e.g., a plasmid) that encodes the antibody.In one embodiment, huCH101 and huCH34 show therapeutic effect as an antibody drug against hepatocellular carcinoma. [Brief description of the drawings]

[0007] [Figure 1] Strategy for humanizing CH101 by CDR grafting. [Diagram 2] Amino acid sequence alignment to compare the variable regions of mouse CH101, germline sequence, and humanized CH101 after CDR grafting. The amino acid sequences of CH101 or germline variable kappa (VK) (Figure 2A) and heavy chain (VH) (Figure 2B) are displayed. Amino acids in the framework regions are colored black and CDR regions are colored red. Amino acids that differ in the framework regions of the germline sequence are colored green and mutations in the framework + CDR regions of the parent CH101 are underlined. [Diagram 3]FACS staining of Δ42PD1 with humanized CH101 antibody panel. A panel of humanized CH101 antibodies was used to distinguish Δ42PD1 from PD1 on the surface of 293T cells by flow cytometry. Different antibody binding activity was color coded as light blue for 293T-Δ42PD1 cells, orange for 293T-PD1 cells, and red for 293T cells. Parental mouse CH101 was used as a positive control for 293T-Δ42PD1 binding. Human IgG1 isotype was used as a negative control. Anti-human CD279(PD-1)-APC was used as a positive control for 293T-PD1 binding. [Figure 4] Assessment of humanized CH101 antibody half-life in vivo. Animal schedule for evaluating antibody half-life (Figure 4A). The in vivo kinetics of CH101-huIgG1 (Figure 4B), CH101-IgG4 (Figure 4C), and hufrCH101-IgG1 (Figure 4D) were measured by ELISA and analyzed. The half-life of the individual antibodies is indicated according to the analysis. [Diagram 5] In vivo evaluation of immunotherapeutic effect of CH101-huIgG1 (chimera) in HCC model mice. (Figure 5A) Experimental schedule. (Figure 5B) Huh7 tumors of each animal at the end point. (Figure 5C) Time course of tumor growth was recorded. (Figure 5D) Tumor weights of each animal at the end point were measured and compared between groups. (Figures 5E-5G) Plasma inflammatory cytokine profiles were measured at the end point (2 animals in the CH101-huIgG1 group and 1 animal in the mouse CH101 group were excluded because they were not evaluable). Statistical analysis was performed using standard one-way ANOVA as well as multiple comparison tests. *p<0.05; **p<0.01, ***p<0.001. [Figure 6]In vivo evaluation of hufrCH101-huIgG1 for immunotherapeutic efficacy in HCC mouse model. (Figure 6A) Experimental schedule. (Figure 6B) Images of Huh7 tumors taken from each animal at the endpoint. (Figure 6C) Time course of tumor growth was recorded weekly. (Figure 6D) Tumor weight was measured for each animal at the endpoint. (Figure 6E-6G) Plasma inflammatory cytokine profile was measured at the endpoint. Statistical analysis was performed using standard one-way ANOVA as well as multiple comparison test. *, p<0.05; **, p<0.01, ***, p<0.001. [Figure 7] High expression of Δ42PD-1 in different T cell subsets in HCC patients. (Fig. 7A-7B) Representative dot plots and quantification results (mean ± SEM) of Δ42PD-1+T cells and PD-1+T cells in PBMCs of 38 HCC patients and 43 healthy controls. (Fig. 7C-7D) Representative dot plots and quantification results (mean ± SEM) of Δ42PD-1+CD4+T cells and Δ42PD-1+CD8+T cells in PBMCs of these HCC patients. (Fig. 7E-7F) Representative dot plots and quantification results (mean ± SEM) of Δ42PD-1+T cells or PD-1+T cells in tumor tissues and adjacent non-tumor tissues from 25 HCC patients. (Fig. 7G) Frequency of Δ42PD-1+CD4+T cells or Δ42PD-1+CD8+T cells in tumor tissues and adjacent non-tumor tissues from the same patient (n=25). (Fig. 7H) Correlation between the frequency of Δ42PD-1+ T cells and PD-1+ T cells in PBMCs (n=38) and tumors (n=25), respectively, in the same cohort of HCC patients. (Fig. 7I) Frequency (mean ± SEM) of PD-1+ T cells and Δ42PD-1+ T cells in PBMCs and tumors of HCC patients stratified into UICC8 stage groups (PBMCs: stage I (n=15), stage II (n=11), and stage III (n=9) and tumors: stage I (n=7), stage II (n=9), and stage III (n=7)). p<0.05 was considered significant: *p<0.05, **p<0.01, and ***p<0.001. [Figure 8]Anti-Δ42PD-1 antibody suppresses HCC development in mouse models. (Figure 8A) Growth curves of Huh7 and PLC5 in NSG-huPBL mice (n=6 / group). (Figures 8B-8C) Representative dot plots and frequency of PD-1+Δ42PD1- T cells and PD-1+Δ42PD-1+ T cells among total human CD45+CD3+ T cells in the spleen or TME were measured in NSG-huPBL and Huh7-NSG-huPBL mice. (Figure 8D) Schematic of treatment schedule. Huh7 cells were inoculated subcutaneously, grown for 1 week, and intraperitoneally injected daily with 200 μg of CH101, anti-PD-1 antibody (nivolumab), or isotype, respectively, for 5 doses. (Figure 8E) Tumor growth curves of Huh7-NSG-HuPBL mice after CH101, anti-PD-1 nivolumab, or isotype treatment. Results shown are representative of two independent experiments with consistent findings (n=4 / group). (Figure 8F) Tumor growth curves (mean ± SEM) were measured for Huh7 in NSG mice, or Huh7-TLR4KO in NSG-HuPBL mice (n=4 / group). (Figures 8G-8H) Orthotopic MHCC-97L tumor growth was measured by bioluminescence imaging and expressed as mean S / S0 values ​​(H). Here, S is the bioluminescence signal on the day of evaluation, and S0 is the bioluminescence signal before treatment (n=9 / group). Results are cumulative data from two independent experiments. (Figure 8I) One representative liver image at the endpoint is shown. Arrows indicate intrahepatic tumors. (Figure 8J) Spearman's correlation of Δ42PD-1+ T cells and IL-6 intensity in HCC patients. Dashed lines indicate linear regression. (Fig. 8K) Representative images (40x) of tumor samples from one patient showed colocalization of Δ42PD-1+ TIL cells and IL-6-producing TLR4+ HCC cells. (Fig. 8L) Quantification of Δ42PD-1+ T cells and PD-1+ T cells in PBMCs, spleens, and tumors of Huh7-NSG-huPBL mice at endpoint (mean ± SEM). (Fig. 8M) Quantification of IL-6 and IL-8 in plasma at endpoint (mean ± SEM). (Fig. 8N) Representative immunofluorescence staining (×20) in tumor tissues from Huh7-NSG-huPBL mice is shown with color coding of cellular expression of IL-6, TLR4, and CD3, respectively.p<0.05 was considered significant: *p<0.05, **p<0.01, and ***p<0.001. [Figure 9] Light and heavy chain sequences of the mouse Δ42PD-1 specific CH34 monoclonal antibody and the CDR regions essential for generating the humanized monoclonal antibody, color coded. [Figure 10] Sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the humanized Δ42PD-1-specific CH34 monoclonal antibody, with the CDR regions essential for the generation of the humanized monoclonal antibody color-coded. [Figure 11] Specificity of the cloned antibody (top) and hybridoma-derived anti-Δ42PD1 antibody CH34, which binds to Δ42PD1 but not to PD-1 expressed on human 293T cells. [Figure 12] Specificity of the cloned anti-Δ42PD1 antibody CH34, which binds to Δ42PD1 but not to PD-1 expressed on human B cells. [Figure 13]Increased Δ42PD-1 expression is accompanied by B cell exhaustion in CHP. (Figure 13A) Representative flow cytometry analysis of CD19+ B cells from BD (n=14) and untreated CHP (n=42). Numbers represent percentages of gated populations. (Figure 13B) Summary of data from Figure 13A showing mean fluorescence intensity (MFI) of Δ42PD-1 and frequency of Δ42PD-1+ B cells in BD and CHP. (Figure 13C) Color-coded gating on CD21 / CD27 stained B cells from representative BDs and CHPs to identify CD21-CD27+ activated memory (AM), CD21-CD27+ resting memory (RM), CD27-CD21- tissue-like memory and anergic naive B cells (TLM / aN), and CD27-CD21+ resting naive plus intermediate memory B cells (IM / rN). (Figure 13D) Summary of data from Figure 13C, showing increased frequency of AM and TLM / aN B cells in CHPs compared to BDs. (Figure 13E) Δ42PD-1 expression in color-coded AM, TLM / aN, RM, and IM / rN B cells from representative BDs and CHPs. Numbers indicate MFI of Δ42PD-1 on cells. (FIG. 13F) Summary of data from FIG. 13E showing MFI of Δ42PD-1 in B cell subsets from BD and CHP. (FIG. 13G) Representative flow cytometry analysis of PD-1+ and Δ42PD-1+ AM, TLM / aN, RM, and IM / rN B cells from BD and CHP. (FIG. 13H) Summary of data from FIG. 13G showing frequency of Δ42PD-1+ AM, TLM / aN, RM, and IM / rN B cells in BD and CHP. Data represent a single measurement for each sample. Each dot in FIG. 13B, FIG. 13D, FIG. 13F, and FIG. 13H represents one individual.Statistical significance was determined using common one-way ANOVA and Benjamini, Krieger and Yekutieli's two-step linear step-up procedure and single pooled variance for Figure 13B, unpaired t-test for Figure 13D, and common two-way ANOVA and Benjamini, Krieger and Yekutieli's two-step linear step-up procedure and single pooled variance for Figures 13F and 13H. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, and ****, P ≤ 0.0001. Average data are presented as mean ± SEM. [Figure 14]Anti-Δ42PD-1 antibodies decrease SHP1 recruitment and promote the AKT1 / FOXO1 pathway and B cell proliferation. (Figure 14A) Representative flow cytometry analysis. (Figure 14B) Summary of data in Figure 14A. Primary B cells (n=6) from BD are prelabeled with FITC-CFSE and stimulated with α-IgM / GF(ab')2 (10 μg / ml) and msIgG2b control antibody or CH34. Cells are analyzed by flow cytometry on day 5. (Figure 14C) CH34 decreases SHP1 recruitment by Δ42PD-1. Primary B cells from BD are stimulated with α-IgM / GF(ab')2 (10 μg / ml) and msIgG2b control antibody or CH34, lysed on day 2, and subjected to immunoprecipitation and western blotting with antibodies against Δ42PD-1 and SHP1, respectively. (Figure 14D) CH34 treatment expanded the Ki67+ B cell population. (Figure 14E) CH34 treatment increased phoph-ACT1-S473 and phosph-FOXO1-S256 levels and decreased P27 protein expression. (Figure 14F) CH34 treatment increased FOXO1 and P27 mRNA in B cells (n=3). In Figure 14D-F, primary B cells were cultured in complete RPMI1640 medium supplemented with α-IgM / GF(ab')2 (10 μg / ml) and msIgG2b control antibody (10 μg / ml) or CH34 (10 μg / ml), or SHP1 inhibitor NSC87877 (10 uM). After 48 hours, cells were stained for flow cytometry analysis (Figure 14D), or lysed for Western blotting detection of AKT1, FOXO1, and P27 (Figure 14E), or qPCR of FOXO1 and P27 mRNA (Figure 14F). Statistical significance was determined using paired t-test. *, p<0.05, and ***, p<0.001. Average data presented as mean ± SEM. [Figure 15]CH34 promotes B cell proliferation after BCR stimulation in HIV patients. (Figure 15A-B) CH34 increases the proliferative response of naive B cells (IgD+) and memory B cells (IgD-) after BCR stimulation (n=20). (Figure 15A) Representative flow cytometry analysis. (Figure 15B) Summary of data from Figure 15A. (Figure 15C-D) CH34 increases CD86 expression in CHP of naive and memory B cells upon BCR stimulation (n=20). (Figure 15C) Representative flow cytometry analysis. (Figure 15D) Summary of data from Figure 15C. (Figure 15E-F) CH34 increases the proliferative response of B cells from CHP after TD stimulation (n=10). (Figure 15E) Representative flow cytometry analysis. (Figure 15F) Summary of data from Figure 15E. (Figure 15G-H) CH34 increases the proliferative response of naive B cells from CHP after TI stimulation (n=10). (Figure 15G) Representative flow cytometry analysis. (Figure 15H) Summary of data in Figure 15G. IgD+ and IgD- B cells isolated from PBMCs of CHP were prelabeled with CFSE and cultured in RPMI1640 medium supplemented with α-IgM / GF(ab')2 (10 μg / ml), TD, or TI stimulation + CH34 (10 μg / ml), or msIgG2b control (10 μg / ml) for 5 days and analyzed by flow cytometry. Statistical significance was determined using common one-way ANOVA and the Benjamini, Krieger and Yekutieli two-step linear step-up procedure with single pool variance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, and ****, P ≤ 0.0001. Average data are presented as mean ± SEM. [Figure 16] B cells express high levels of Δ42PD-1 after BCR stimulation and upon HIV-1 infection. Δ42PD-1 inhibits BCR-triggered cell cycle progression through recruitment of SHP1. SHP1 binds to AKT1 and inhibits AKT1 activation, suppressing the AKT1 / FOXO1 pathway. Δ42PD-1-specific antibodies restore proliferation of B cells in HIV-1 patients.

[0008] A brief description of the sequence SEQ ID NO:1: CH101 VH amino acid sequence SEQ ID NO:2: CH101 VK amino acid sequence SEQ ID NO: 3: hufrCH101 VH amino acid sequence SEQ ID NO: 4: hufrCH101 VK amino acid sequence SEQ ID NO:5: Nucleotide sequence encoding CH101 VH SEQ ID NO:6: Nucleotide sequence encoding CH101 VK SEQ ID NO: 7: Nucleotide sequence encoding hufrCH101 VH SEQ ID NO: 8: Nucleotide sequence encoding hufrCH101 VK SEQ ID NO: 9: Human germline variable heavy chain amino acid sequence SEQ ID NO: 10: Human germline variable kappa chain amino acid sequence SEQ ID NO: 11: Human heavy chain J region amino acid sequence SEQ ID NO: 12: Human kappa chain J region amino acid sequence SEQ ID NO: 13: Nucleotide sequence encoding human germline variable heavy chain SEQ ID NO: 14: Nucleotide sequence encoding human germline variable kappa chain SEQ ID NO: 15: Nucleotide sequence encoding the human heavy chain J region SEQ ID NO: 16: Nucleotide sequence encoding the human kappa chain J region SEQ ID NO: 17: Human kappa chain constant fragment nucleic acid sequence (IgG1 and IgG4) SEQ ID NO: 18: Human kappa chain constant fragment amino acid sequence (IgG1 and IgG4) SEQ ID NO: 19: Human heavy chain constant fragment amino acid sequence (IgG1) SEQ ID NO: 20: Human heavy chain constant fragment nucleic acid sequence (IgG1) SEQ ID NO: 21: Human heavy chain constant fragment amino acid sequence (IgG4) SEQ ID NO: 22: Human heavy chain constant fragment nucleic acid sequence (IgG4) SEQ ID NO: 23: CH101 VH CDR1 amino acid sequence SEQ ID NO:24: CH101 VH CDR2 amino acid sequence SEQ ID NO: 25: CH101 VH CDR3 amino acid sequence SEQ ID NO: 26: CH101 VL CDR1 amino acid sequence SEQ ID NO: 27: CH101 VL CDR2 amino acid sequence SEQ ID NO: 28: CH101 VL CDR3 amino acid sequence SEQ ID NO:29: CH34 VH CDR1 amino acid sequence SEQ ID NO: 30: CH34 VH CDR2 amino acid sequence SEQ ID NO: 31: CH34 VH CDR3 amino acid sequence SEQ ID NO: 32: CH34 VL CDR1 amino acid sequence SEQ ID NO: 33: CH34 VL CDR2 amino acid sequence SEQ ID NO: 34: CH34 VL CDR3 amino acid sequence SEQ ID NO: 35: Mouse CH34 VH amino acid sequence SEQ ID NO: 36: Mouse CH34 VL amino acid sequence SEQ ID NO: 37: Humanized CH34 VH amino acid sequence SEQ ID NO: 38: Humanized CH34 VL amino acid sequence SEQ ID NO: 39: Signal peptide sequence SEQ ID NO: 40: Mouse CH34 heavy chain amino acid sequence SEQ ID NO: 41: Mouse CH34 kappa chain amino acid sequence DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Additionally, to the extent that "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a similar manner to the term "comprising." The transitional terms / phrases (and grammatical variations thereof) "comprising," "comprises," "comprise," "consisting essentially of," "consists essentially of," "consisting," and "consists" can be used interchangeably.

[0010] The phrases "consisting essentially of" or "consists essentially of" indicate that the claim includes embodiments that include the specified materials or steps, and that do not materially affect the basic and novel feature(s) of the claim.

[0011] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method of measurement of the value, i.e., the limitations of the measurement system. In the context of compositions containing amounts of ingredients where the term "about" is used, these compositions contain the stated amounts of ingredients with a 0-10% variation (X±10%) (error range) around the value. In other contexts, the term "about" provides a 0-10% variation (X±10%) (error range) around the given value. As is evident, this variation indicates a range of up to 10% above and below the given value, for example, X±1%, X±2%, X±3%, X±4%, X±5%, X±6%, X±7%, X±8%, X±9%, or X±10%.

[0012] In this disclosure, ranges are described in shorthand notation to avoid redundantly describing each value within the range. Where appropriate, any suitable value within the range can be selected as the upper, lower, or end value of the range. For example, the range 0.1-1.0 indicates end values ​​of 0.1 and 1.0, as well as intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges subsumed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0. Values ​​with at least two significant digits within the range are contemplated, for example, the range 5-10 indicates all values ​​between 5.0 and 10.0, including the end values, as well as between 5.00 and 10.00. When ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed ranges) and certain embodiments therein are expressly included.

[0013] As used herein, the term "subject" refers to an animal that requires or desires the delivery of the benefit provided by the therapeutic composition. The animal may be, for example, a human, a pig, a horse, a goat, a cat, a mouse, a rat, a dog, an ape, a fish, a chimpanzee, an orangutan, a guinea pig, a hamster, a cow, a sheep, a bird, a chicken, as well as vertebrates and invertebrates. These benefits include, but are not limited to, treatment of a condition, disease, or disorder; prevention of a condition, disease, or disorder; immune health; enhancement of enamel, organs, tissues, or body system function. A preferred subject in the context of the present invention is a human. The subject may be of any age or stage of development, including infants, toddlers, adolescents, adults, and the elderly.

[0014] As used herein, the terms "therapeutically-effective amount," "therapeutically-effective dose," "effective amount," and "effective dose" are used to mean an amount or dose of an antibody or composition that, when administered to a subject, is capable of treating or ameliorating a condition, disease, or disorder of the subject, or providing enhanced health or function to an organ, tissue, or body system. In other words, when administered to a subject, the amount is "therapeutically effective." The actual amount administered will vary depending on many factors, including, but not limited to, the particular condition, disease, or disorder being treated or ameliorated; the severity of the condition; the particular organ, tissue, or body system whose health or function is desired to be improved; the patient's weight, height, age, and health; and the route of administration.

[0015] As used herein, the term "treatment" refers to eradicating, alleviating, ameliorating, or reversing to any extent the signs or symptoms of a condition, disease, or disorder, including, but not necessarily, a complete cure of the condition, disease, or disorder. Treatment refers to curing, ameliorating, or partially ameliorating a disorder. "Treatment" also includes improving or enhancing a condition or characteristic, such as, for example, causing the function of a particular system in the body to be in a healthy or homeostatic state.

[0016] As used herein, "prevention" of a condition, disease, or disorder means avoiding, delaying, preventing, or minimizing the onset of a particular sign or symptom of that condition, disease, or disorder, i.e., the sign or symptom may develop at a later date. Prevention can include reducing the severity of the onset of such condition, disease, or disorder and / or inhibiting the progression of the condition, disease, or disorder to a more severe condition, disease, or disorder.

[0017] By "reduces" it is meant a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0018] By "increases" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0019] In some embodiments of the invention, the method includes administration of multiple doses of the antibody composition of the subject invention. The method can include administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeutically effective amounts of a composition comprising an antibody of the subject invention described herein. In some embodiments, the doses are administered for a period of 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, 30, or more than 30 days. Furthermore, treatment of a subject with a therapeutically effective amount of an antibody of the invention can include one treatment or a series of treatments. It will also be understood that the effective amount of the antibody used for treatment can increase or decrease over the course of a particular treatment. The variation in dosage can result from and be evident from the results of diagnostic assays or imaging techniques for detecting tumor size as known in the art. In some embodiments of the invention, the method includes administration of the antibody multiple times per day, including but not limited to twice a day, three times a day, and four times a day.

[0020] The present disclosure includes all amino acid sequences described herein and all polynucleotides encoding the amino acid sequences. The present disclosure also includes all amino acid sequences that have at least 80% similarity to the amino acid sequences described herein. Percent amino acid sequence can be determined over the entire length of any amino acid sequence described herein or over any contiguous amino acid sequence that constitutes a functional antibody such as huCH101, non-limiting examples of which are shown below. Thus, the present disclosure includes sequences that are identical to any amino acid or nucleotide sequence described herein, and sequences that are 80 to 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to such sequences.

[0021] The term "humanized" as used herein refers to antibodies from non-human species, such as mouse, that have been modified to resemble more closely the antibody variants naturally produced in humans.

[0022] The recitation of a list of chemical groups in a definition of a variable herein includes that variable being defined as a single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as a single embodiment or in combination with other embodiments or portions thereof.

[0023] Humanized Δ42PD1-specific mAb The present invention provides antibodies targeting an alternatively spliced ​​isoform of PD-1 (referred to herein as "Δ42PD1"). In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the variable regions, including the heavy and / or light chain variable regions, of the antibody targeting Δ42PD1 can be linked to a human antibody constant fragment, including, for example, a human IgG4 constant fragment (SEQ ID NO:21), preferably a human IgG1 constant fragment (SEQ ID NO:19). In an embodiment, the antibody targeting Δ42PD1 is mouse CH101 or mouse CH34. CH101 and CH34 have been previously described: (See U.S. Pat. No. 10,047,137 and U.S. Patent Application 16 / 030,180, which are incorporated by reference in their entireties). The complementarity determining regions of the antibody of the subject invention can be derived from mouse CH101 or CH34 hybridoma and the framework regions are derived from human germline.

[0024] The variable region of CH101 can be linked with human IgG1 constant fragment, and the constructed chimeric CH101 can show a humanization rate of about 66.1% (437 / 661). Optionally, the antibody can be further humanized. Specifically, the variable region of chimeric CH101 can be modified to increase the humanized portion to about 97.0% (641 / 661), resulting in a humanized CH101 antibody (huCH101). The variable region of CH34 can be linked with a human constant fragment, such as a human IgG1 or IgG4 constant fragment and / or a human kappa constant fragment. In some embodiments, complementarity determining region (CDR) grafting can be utilized to alleviate immunogenicity. The approach to perform CDR grafting is to first determine the CDR region of a non-human antibody. Secondly, the most homologous one suitable as a receptor can be searched from the human germline. Finally, the mouse CDR can be grafted into the human framework region (FR).

[0025] Accordingly, the present disclosure provides a Δ42PD1 specific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), (i) VH comprises HCDRs 1 to 3 having the amino acid sequences shown in SEQ ID NOs: 23 to 25, respectively, and VL comprises HCDRs 1 to 3 having the amino acid sequences shown in SEQ ID NOs: 26 to 28, respectively; or (ii) VH comprises HCDR1 to 3 having the amino acid sequences shown in SEQ ID NOs: 29 to 31, respectively, and VL comprises LCDR1 to 3 having the amino acid sequences shown in SEQ ID NOs: 32 to 34, respectively; A Δ42PD1-specific antibody or antigen-binding fragment thereof is provided.

[0026] Although the present disclosure can use CDRs defined according to either of these numbering systems, preferred embodiments use the Kabat or Chothia defined CDRs.

[0027] When CDR sequences are defined according to the Kabat numbering system, the VH of the antibodies disclosed herein comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:23 (GYSITSGF), SEQ ID NO:24 (HYSGS), and SEQ ID NO:25 (SDF), respectively, and the VL of the antibodies disclosed herein comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:26 (TSSQSLLDRDGETFMN), SEQ ID NO:27 (LVSKLDF), and SEQ ID NO:28 (WQGTHFPL), respectively; or The VH of the antibody disclosed herein comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO:29 (GFTFSDYG), SEQ ID NO:30 (ISSLAYTI), and SEQ ID NO:31 (ARGGAGFAY), respectively, and the VL of the antibody disclosed herein comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:32 (DHINNW), SEQ ID NO:33 (GAT), and SEQ ID NO:34 (QQYWSTPWT), respectively.

[0028] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2; or

[0029] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4.

[0030] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:35, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:36.

[0031] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:37, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:38.

[0032] In a preferred embodiment, the VH comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 2. In another preferred embodiment, the VH comprises the amino acid sequence set forth in SEQ ID NO: 3 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 4. In another preferred embodiment, the VH comprises the amino acid sequence set forth in SEQ ID NO: 35 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 36. In another preferred embodiment, the VH comprises the amino acid sequence set forth in SEQ ID NO: 37 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0033] In some embodiments, the antibody is an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0034] In some embodiments, the antibody comprises a heavy chain constant region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19 or 21. In some embodiments, the antibody comprises a light chain constant region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.

[0035] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, ds-scFv, and dAb.

[0036] In some embodiments, the antibody is a murine, human, or humanized antibody. In preferred embodiments, the antibody is a humanized antibody.

[0037] In some embodiments, the antibody is a monoclonal antibody.

[0038] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:40, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:41.

[0039] In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody that further comprises a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen, such as a T cell antigen.

[0040] In an embodiment, the humanized Δ42PD1 specific antibody comprises a variable heavy chain having SEQ ID NO:3 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO:3, and a variable light chain having SEQ ID NO:4 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO:4. In an embodiment, the light chain is a kappa light chain. In an embodiment, the humanized Δ42PD1 specific mAb comprises a human IgG1 constant heavy chain fragment according to SEQ ID NO:19 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:19, and a human IgG1 light chain fragment according to SEQ ID NO:18 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:18.

[0041] In an embodiment, the humanized Δ42PD1 specific antibody comprises a variable heavy chain encoded by SEQ ID NO: 7 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7, and a variable light chain encoded by SEQ ID NO: 8 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In an embodiment, the light chain is a kappa light chain. In an embodiment, the humanized Δ42PD1 specific mAb comprises a human IgG1 constant heavy chain fragment encoded by SEQ ID NO:20 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO:20, and a human IgG1 light chain fragment encoded by SEQ ID NO:17 or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO:17.

[0042] Either the heavy or light chain variable region contains three CDRs and four FRs. CDRs are the main contact regions with the antigen and retain the main functions of the antibody. In one embodiment, the percentage of sequence mutations in the FR region, for example according to SEQ ID NO: 9 and / or SEQ ID NO: 10, can reach about 10% and retain the ability to bind to Δ42PD1. For humanized CH101, all CDRs of parent CH101 were retained and functional changes after humanization were tested. As a result, hufrCH101 retained 76.79% of the function of parent mouse CH101 (Table 1). The average sensitivity of hufrCH101-IgG1 to 293T-Δ42PD1 cells is about 45.46%, and the average sensitivity of parent mouse CH101 to 293T-Δ42PD1 cells is about 59.2%. This association can be interpreted as hufrCH101-IgG1 retaining about 76.79% of the function of parent mouse CH101.

[0043] In a preferred embodiment, compositions and methods according to the subject invention utilize a humanized Δ42PD1-specific mAb. The humanized Δ42PD1-specific mAb may be added to the composition at a concentration of 0.01-90% by weight (wt%), preferably 0.1-50 wt%, and more preferably 0.1-20 wt%. In another embodiment, the purified humanized Δ42PD1-specific mAb may be combined with an acceptable carrier, where the humanized Δ42PD1-specific mAb may be present at a concentration of 0.001-50% (v / v), preferably 0.01-20% (v / v), and more preferably 0.02-10% (v / v). In one embodiment, the humanized Δ42PD1-specific mAb may be administered to the subject at a dose of about 1 mg / kg to about 100 mg / kg.

[0044] In one embodiment, the subject compositions are formulated as orally ingestible products, such as food, capsules, tablets, or drinkable liquids. Orally deliverable pharmaceuticals are physiologically active substances that are delivered via initial absorption into the gastrointestinal tract or oral mucosa. Topical compositions can also be formulated as solutions that can be administered via injection, including, for example, intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous. In other embodiments, the subject compositions are formulated to be administered through the skin through a patch or directly onto the skin for local or systemic effect. The compositions can be administered sublingually, bucally, rectally, or vaginally. Additionally, the compositions can be sprayed into the nose and absorbed through the nasal membrane, atomized, inhaled through the mouth or nose, or administered to the eyes or ears.

[0045] An orally consumable product according to the invention is a preparation or composition suitable for ingestion, nutrition, oral hygiene, or palatability, which is intended to be introduced into the oral cavity of a human or animal, remain there for a period of time, and then be swallowed (e.g., an easy-to-ingest food or tablet) or removed again from the oral cavity (e.g., a chewing gum, or an oral hygiene product, or a medical mouthwash). Although an orally deliverable pharmaceutical agent can be formulated into an orally ingestible product, and an orally ingestible product can contain an orally deliverable pharmaceutical agent, the two terms are not intended to be used interchangeably herein.

[0046] Consumable products include any substance or product that is intended to be consumed by humans or animals in a processed, semi-processed or raw state, and also includes substances that are added during the manufacturing, treatment or processing of ingestible products, particularly foods and medicines, and that are intended to be introduced into the oral cavity of humans or animals.

[0047] An orally ingestible product may also include material that is intended to be swallowed by a human or animal and subsequently digested, either intact, prepared, or processed. Thus, an orally ingestible product according to the present invention also includes casings, coatings, or other encapsulants that are intended or anticipated to be swallowed along with the product.

[0048] In one embodiment, the orally consumable product is a capsule, tablet, syrup, emulsion, or liquid suspension containing the desired orally deliverable substance, hi one embodiment, the orally consumable product can include the orally deliverable substance in a powder form, which can be mixed with water or other liquid to produce a drinkable, orally consumable product.

[0049] In some embodiments, an orally consumable product according to the present invention may include one or more formulations for nutritional or palatable purposes. These include in particular bakery products (e.g. bread, dry biscuits, cakes and other pastries), confectionery (e.g. chocolate, chocolate bar products, other bar products, fruit gums, coated tablets, hard caramels, toffee and caramel, and chewing gum), alcoholic or non-alcoholic beverages (e.g. cocoa, coffee, green tea, black tea, black tea or green tea drinks concentrated with green tea or black tea extracts, rooibos tea, other herbal teas, fruit-containing lemonades, isotonic drinks, soft drinks, nectars, fruit or vegetable juices, and fruit or vegetable juice preparations), instant drinks (e.g. instant cocoa drinks, instant tea drinks, instant coffee drinks, etc.), meat products (e.g. ham, raw or raw sausage preparations, seasoned or marinated raw or cured meat products), eggs or egg products (e.g. dried whole eggs, egg whites, egg yolks), cereal products (e.g. breakfast cereals, muesli bars, cooked instant rice products), dairy products (e.g. full-fat or low-fat fat and non-fat milk drinks, rice pudding, yogurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, and partially or fully hydrolyzed products containing milk proteins), products based on soy protein or other soy fractions (e.g. soy milk and preparations thereof, beverages containing isolated or enzymatically treated soy protein, beverages containing soy flour, preparations containing soy lecithin, fermented products such as tofu or tempeh, preparations thereof, and fruit preparations and mixtures, optionally with flavouring substances), fruit preparations (e.g. jams, fruit ice cream, fruit sauces, and fruit fillings), vegetable preparations (e.g. ketchup, sauces, dried, frozen, cooked, and boiled vegetables), snack foods (e.g. baked or fried potato chips (crisps), potato dough products, and extruded products based on corn or peanuts), products based on fats and oils or emulsions thereof (e.g. mayonnaise, remoulade, and dressings),Other prepared foods and soups (e.g. dry soups, instant soups, prepared soups), seasonings (e.g. sprinkle seasonings), sweetener compositions (e.g. tablets, sachets, and other preparations for sweetening or whitening beverages or other foods). The compositions of the present invention may also serve as semi-finished products for the manufacture of other compositions for nutritional or palatable purposes.

[0050] The subject compositions may further comprise one or more pharma- ceutical acceptable carriers and / or excipients, and may be formulated into solid, semi-solid, liquid, or gaseous formulations, such as, for example, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0051] As used herein, the term "pharmaceutical acceptable" means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0052] Carriers and / or excipients according to the subject invention include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl buffer, acetate buffer, phosphate buffer, etc.), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents, e.g., suitable for intravenous use, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, vehicles, bulking agents, chelating agents (e.g., EDT, A or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavors, fragrances, thickeners (e.g., carbomer, gelatin, or sodium alginate), coating agents, preservatives (e.g., thimerosal, benzyl alcohol, polyquaterniums), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tonics, absorption retarders, adjuvants, bulking agents (e.g., lactose, mannitol). The use of carriers and / or excipients in the pharmaceutical and supplement fields is well known. Except for conventional media or agents that are incompatible with the subject health-promoting substance or composition, the use of carriers or excipients in the subject compositions is contemplated.

[0053] In one embodiment, the compositions of the subject invention can be made into an aerosol formulation, e.g., so that they can be nebulized or inhaled. Pharmaceutical formulations suitable for administration in the form of an aerosol or spray are, for example, powders, particles, solutions, suspensions, or emulsions. Formulations for oral or nasal aerosol or inhalation administration can also be formulated with carriers, such as, for example, saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in admixture with powder dispersants or fluorocarbons. Aerosol formulations can be placed in pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Illustratively, delivery can be by use of a single-use delivery device, mist spray, breath-activated powder inhaler, aerosol metered-dose inhaler (MDI), or any other of the many nebulizing delivery devices available in the art. Additionally, direct administration via a mist tent or endotracheal tube is also performed.

[0054] In one embodiment, the subject compositions can be formulated for administration via injection, for example, as a solution or suspension. The solution or suspension can include a suitable non-toxic parenterally acceptable diluent or solvent, such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or suitable dispersing or wetting agents and suspending agents, such as sterile non-irritating fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. Exemplary carriers for intravenous use include mixtures of 10% USP ethanol, 40% USP propylene glycol, or polyethylene glycol 600, and the balance USP Water for Injection (WFI). Other exemplary carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyldiphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral water-in-vegetable oil emulsions. Water or aqueous solutions of saline, dextrose and glycerol are preferably used as carriers, particularly for injectable solutions. Exemplary carriers for subcutaneous or intramuscular use include acceptable isotonic solutions such as phosphate buffered saline (PBS) solutions, 5% dextrose in WFI, 5% dextrose in USP WFI, 0.01%-0.1% triethanolamine in 5% dextrose, or 0.9% sodium chloride in USP WFI, or mixtures of 1:2 or 1:4 USP ethanol, 40% propylene glycol and 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyldiphosphatidylcholine in USP WFI and 1-10% squalene or parenteral water-in-vegetable oil emulsions.

[0055] In one embodiment, the compositions of the subject invention can be formulated for administration via topical application to the skin, for example, as a topical composition, including the use of rinses, sprays, or drops, lotions, gels, ointments, creams, foams, powders, solids, sponges, tapes, vapors, pastes, tinctures, or transdermal patches. Suitable formulations for topical application can include, in addition to any pharma- ceutically active carrier, an emollient, such as, for example, carnauba wax, cetyl alcohol, cetyl esters wax, emulsifying wax, hydrous lanolin, lanolin, lanolin alcohol, microcrystalline wax, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. Additionally, the compositions can include humectants, such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6 hexanetriol, or penetration enhancers, such as ethanol, isopropyl alcohol, and oleic acid.

[0056] Administration of humanized Δ42PD1-specific mAb In certain embodiments, a humanized Δ42PD1-specific mAb can be administered to a subject. Any means of administration that can contact the inhibitor with cells in a subject is contemplated in the subject method, including, for example, oral, intravenous, intraperitoneal, intramuscular, intradermal, or subcutaneous administration. In a preferred embodiment, a humanized Δ42PD1-specific mAb can be administered intravenously.

[0057] In an embodiment, the humanized Δ42PD1-specific mAb can contact healthy cells and / or tumor or cancer cells of a subject. In an embodiment, the humanized Δ42PD1-specific mAb can contact cells of the liver, including cancerous hepatocytes. In an embodiment, the humanized Δ42PD1-specific mAb can inhibit hepatocellular carcinoma and other solid cancers. The humanized Δ42PD1-specific mAb can penetrate cells of a subject, including, for example, cancer cells. In an embodiment, the humanized Δ42PD1-specific mAb can inhibit tumor cell proliferation. The humanized Δ42PD1-specific mAb can inhibit tumor cell proliferation by modulating Δ42PD1 expression and / or the function of the Δ42PD1-TLR4 system.

[0058] Materials and Methods Plasmid construction For expression plasmid construction, the antibody variable heavy chain was assembled in the AbVec2.0-IGHG1 plasmid (Addgene, Watertown, MA) backbone, and the antibody variable kappa chain was assembled in the AbVec1.1-IGKC plasmid (Addgene) backbone using NEBuilder® HiFi DNA Assembly Master Mix (New England BioLabs, Ipswich, MA).

[0059] Antibody detection Mouse plasma was diluted 1:1 with PBS and LEGENDplex (商標) Inflammatory cytokine profiles were measured using the Human Inflammation Panel 1 (13-plex) (Biolegend, San Diego, CA) according to the product instructions. (商標) The assay is a bead-based immunoassay using the same basic principle as sandwich immunoassay. The concentration of a particular analyte is determined using a standard curve made with the same assay (see World Wide Web: biolegend.com / en-us / products / legendplex-human-inflammation-panel-1-13-plex-with-v-bottom-plate-16929?GroupID=GROUP25, which is incorporated herein by reference together with the current manual).

[0060] statistical analysis Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). One-way analysis of variance as well as multiple comparisons were performed to compare group means when more than two groups were involved. Differences between group means within two groups were evaluated only by unpaired t-tests. A p-value <0.05 was considered significant. Specific details regarding statistical tests are provided in the figure legends.

[0061] Patients, procedures, and sample collection Healthy human blood buffy coats with a median age of 31 years (interquartile range 22-67 years; 62.16% male vs. 37.84% female; HBV / HCV negative) were obtained from the Hong Kong Red Cross. Eligible patients were aged 18 years or older, with a diagnosis confirmed by histological or cytological analysis, and clinical characteristics according to the American Association for Liver Diseases. All patients had not received systemic therapy for liver cancer before enrollment. From July 2016 to November 2019, 41 patients who underwent radical resection as standard of care were managed at the Department of Surgery, Queen Mary Hospital, Hong Kong. Samples were collected at two time points: peripheral whole blood was collected before surgical resection, and paired tumor and adjacent nontumor samples were collected after resection.

[0062] Flow cytometry and immunofluorescence analysis Flow cytometric and immunofluorescence analyses were performed and analyzed as previously described. Cells were incubated with Fc Block (Biolegend) in staining buffer (PBS with 2% FBS) for 10 min and then stained with the indicated antibodies (Supplementary Table 2) for 30 min at 4 °C. For intracellular staining, cells were fixed and permeabilized with BD Cytofix / Cytoperm (BD Biosciences) before staining with mAbs in Perm / Wash buffer (BD Biosciences) for 30 min at 4 °C and washed with 1 ml of staining buffer. All samples were acquired using a FACSAria III flow cytometer (BD Biosciences) and results were analyzed using FlowJo software (v10.6) (BD Biosciences).

[0063] The following antibodies were purchased from BioLegend and used for flow cytometry: CD3 (clone HIT3a), CD4 (clone A161A1), CD8a (clone HIT8a), CD45 (clone 2D1), CD19 (clone HIB19), CD16 (clone 3G8), CD14 (clone HCD14), CD11c (clone S-HCL-3), and HLA-DR (clone LN3). ), CD45RA (clone HI100), CCR7 (clone G043H7), CD27 (clone M-T271), IFN-γ (clone B27), TNF-α (MAb11), IL-2 (clone MQ1-17H12), PD-1 (clones EH12.2H7, NAT105, and A17188B), Tim-3 (clone F38-2E2), and LAG3 (clone 11C3C65). The following antibodies were purchased from BD Biosciences and used for flow cytometry: CD56 (NCAM-1), VISTA (clone MIH65), and CD244 (clone 2-69). The Δ42PD-1 antibody (clone CH101) was labeled with Alexa Fluor 568 / 647 using the Antibody Labeling Kit (Invitrogen) as previously described for flow cytometry. Dead cells were stained with the Zombie Aqua Fixable Viability Kit (BioLegend). Cell surface and intracellular immunostaining was performed as previously described. Annexin V staining (BD Bioscience) was performed according to the product manual. Flow cytometry data analysis was performed using FlowJo software (Tree Star, v10).

[0064] Immunofluorescence and immunohistochemistry staining was performed using anti-human CD3 (clone CD3-12, AbD Serotec; #A0452, DAKO), anti-human TLR4 (#ab150583, Abcam), anti-Δ42PD-1 (clone CH101), anti-IL6 (clone M-19, Santa Cruz) and anti-PD-1 (clone NAT105, Abcam) antibodies, with secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 568 or Alexa Fluor 647 (Invitrogen). Counterstaining was performed with Hoechst 33258, and stained sections were mounted with fluorescent mounting medium (DAKO). Images were then acquired using a confocal microscope (LSM710 / 780 / 800, Carl Zeiss, 20x and 40x objectives) with ZEN software. The integrated intensity of the immunostained signal and the cell number were quantified using the software ImageJ.

[0065] Humanized Mouse Models ImmunodeficiencyNOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG, Jackson Laboratories) were bred and housed in the minimal disease area of ​​the HKU Laboratory Animal Unit (LAU). All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of the Laboratory Animal Unit of the University of Hong Kong (License No. 3883-16). Mice of both sexes were randomly selected without blinding. Mice aged 6–8 weeks were inoculated with 1.0–1.5 × 10 7 healthy human PBMCs were injected intraperitoneally in 0.5 ml of PBS. Animals were blooded on day 10 to examine the levels of human lymphocytes by flow cytometry. Erythrocytes in blood samples were lysed with erythrocyte lysis buffer (BD PharmLyse) before staining. Successfully engrafted mice (NSG-huPBL) were used to establish the HCC model. For the subcutaneous injection HCC model, 2 × 10 6 Huh7 or PLC5 cells were injected subcutaneously into the right hind flank of NSG-huPBL mice. Tumor volumes were measured with a caliper (tumor volume = 1 / 2 (length × width)). 2In the orthotopic HCC model, subcutaneously injected MHCC-97L tumors were harvested from NSG mice when they reached a diameter of 1 cm and dissected under aseptic conditions. 3 Intact pieces measuring 100x the size of 10x were grafted into the left hepatic lobe of each NSG-huPBL mouse. Luciferase-expressing tumors were measured with an IVIS Spectrum (PerkinElmer) and expressed as photons / s / cm within a region of interest (ROI) using Living Image software (version 4.0, PerkinElmer) as previously described. 2 Specimens were fixed in formalin-zinc fixative (Sigma) and embedded in paraffin blocks.

[0066] Ex vivo cell preparation Mouse PBMCs and splenocytes were isolated as previously described. Fresh PBMCs were isolated from peripheral blood of healthy donors or HCC patients by gradient centrifugation (Ficoll-Paque, GE Healthcare). Tumor or adjacent non-tumor tissues were cut into fragments and digested under a gentleMACS Octo Dissociator (Miltenyi Biotec) using a Tumor Dissociation Kit (Miltenyi Biotec) according to the product manual. Cells were passed through a 70 μm strainer and then subjected to a 40% / 80% Percoll gradient (Sigma). Interphase leukocytes were collected by centrifugation at 800 g for 20 min. CD3 + T cells were isolated using the Pan T Cell Isolation Kit (Miltenyi Biotech). + T cells and Δ42PD-1 - T cells were isolated using a cell sorter (BD AriaIII) after specific antibody staining.

[0067] In vitro T cell culture, proliferation assay, and cytotoxicity assay Cells were cultured in RPMI1640 (gibco) supplemented with 10% FBS (gibco) and antibiotics at 37 °C and 5% CO2 in 96-well U-bottom plates. For proliferation assays, purified T cells were stained with carboxyfluorescein 6 succinimidyl ester (CFSE, Thermo Scientific) before culture and incubated with a T cell activation / expansion kit (Miltenyi Biotec) at a bead-to-T cell ratio of 1:4 for 4–5 days before analysis by flow cytometry (BD AriaIII). Culture supernatants were harvested to analyze cytokine production. The cytotoxic effect of purified T cells against Huh7 cells was measured using the NonRadioactive Cytotoxicity Assay (Promega) according to the product manual. All cytokine concentrations in plasma, culture supernatants or tumor lysates were measured by LEGENDplex human panel (13-plex, BioLegend). Tumors were minced and homogenized in T-PER tissue protein extraction reagent (Thermo Scientific) supplemented with a protease inhibitor cocktail (Roche) before analysis.

[0068] HIV-1 infected subjects A total of 86 HIV-1-infected patients, including 12 ART patients and 74 treatment-naïve patients, recruited from Shenzhen Third People's Hospital, Shenzhen, Guangdong Province, China, were studied. The study was conducted with the approval of the Hong Kong West Cluster Research Ethics Committee, Hong Kong East Cluster Research Ethics Committee, and Kowloon West Cluster Research Ethics Committee, University of Hong Kong / Hospital Authority (UW 19-834). Blood samples were collected after written informed consent was obtained from each participant. All patients were seronegative for hepatitis B and hepatitis C, and none showed signs of active tuberculosis.

[0069] Isolation of peripheral blood mononuclear cells (PBMCs) PBMCs were isolated from fresh blood samples drawn from patients and healthy donors using Ficoll-Paque density gradient centrifugation in a BSL-3 laboratory. Purified PBMCs were used for immune cell phenotyping and B cell isolation. The remaining cells were frozen at 5 × 10 in freezing medium (90% FBS + 10% DMSO) at -150 °C. 6 The cells were cryopreserved at a density of 100 cells / mL.

[0070] Antibody purification Δ42PD1-specific monoclonal antibodies CH101 and CH34 are purified as previously described (Cheng et al., 2015; Cheung et al., 2017). Briefly, CH34 or CH101-producing hybridoma cells are injected intraperitoneally into 6-8 week-old BALB / c mice primed with incomplete Freund's adjuvant. After 1-2 weeks, ascites are removed from the peritoneal cavity and incubated at 37°C for 1 h and then at 4°C overnight. The ascites are then centrifuged at 4000 x g and 4°C for 30 min and purified using a Pierce (商標) Dilute in 1 to 2 parts PBS to purify the antibody using protein G agarose. The anti-Δ42PD1 antibody CH34 was labeled with Alexa Fluor 647 using the Antibody Labeling Kit (Invitrogen).

[0071] Immunocytochemistry assay Harvest the cells and 6The cells were concentrated to 1000 cells / ml. 10 μl of the cell suspension was dropped onto a clean glass slide and then dried in a hood for 1 h. Samples were fixed in either cold methanol or 10% neutral buffered formalin for 10 min at RT and permeabilized with 0.5% Triton X-100 for 10 min. Samples were then stained with primary antibodies for 1 h at RT, washed with PBS, and stained with fluorescein-conjugated secondary antibodies for 1 h at RT. Cells were treated with Hoechst33258 (Cat. No. H3569, Life Technologies) diluted 1:10,000 for 10 min at RT. After washing, microscopic images were obtained using a Carl Zeiss LSM 780 confocal microscope (Oberkochen, Germany).

[0072] Isolation of B cells Total B cells were isolated from human PBMCs using a pan B cell isolation kit (Miltenyi Biotec) by IgD positive selection with anti-IgD microbeads (Miltenyi Biotec) according to the product manual.

[0073] B cell culture B cells were cultured for 5 days in the presence of α-IgM / GF(ab')2 (10 μg / ml, Thermofisher) alone, α-IgM / GF(ab')2 + CpG (1 μM, Invitrogen), or α-IgM / GF(ab')2 (10 μg / ml) + IL-2 (20 U / ml, R&D Systems), IL-10 (100 ng / ml, R&D Systems), and CD40L (500 ng / ml, PeproTech) in RPMI 1640 medium containing 10% FBS and 1% streptomycin / penicillin (both Gibco). To measure B cell proliferation, B cells labeled with carboxyfluorescein-6-succinimidyl ester (CFSE, 5 μM in PBS, Biolegend) were cultured in 96-well U-bottom plates for 4–5 days. Proliferating B cells were determined by calculating the percentage of low CFSE cells. For immunoprecipitation / Co-IP and Western blot assays, B cells were cultured for 2 days. For CH34-treated experiments, 10 μg / mL CH34 or isotype control msIgG2b was added to B cells on day 0.

[0074] Immunoprecipitation / Co-IP and Western blot assays For cell lysis, 1% SDS lysis buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 5 mM DTT, and 1% SDS) containing protease and phosphatase inhibitors (Thermo Fisher Scientific) was used. The prepared protein lysate samples were incubated with the relevant immunoprecipitating antibodies overnight at 4 °C with shaking, then incubated with protein G agarose beads and further shaken for 3–4 h. The precipitates were washed 4–5 times with lysis buffer. Finally, SDS loading buffer was added, boiled for 5 min, and subjected to SDS-PAGE electrophoresis. The immunoprecipitates or protein lysates were separated by SDS-PAGE using 8% or 12% polyacrylamide gels (80 V, ~30 min; 120 V, ~1.2 h) and transferred to polyvinylidene difluoride (PVDF) membranes (0.3 A, 1.2 h). The transferred membrane was then blocked with 5% skim milk (dissolved in TBST) for 1 hour at room temperature. After blocking, it was incubated with primary antibody solution (diluted 1 / 1000 in 5% BSA TBST buffer) overnight at 4°C. The membrane was then washed with TBST for 3×10 minutes. After washing, the membrane was incubated with fluorescently labeled secondary antibody for 1.5 hours at room temperature with shaking. The membrane was then washed again with TBST for 3×10 minutes. The membrane was then dried and scanned with Li-COR Odyssey.

[0075] RNA isolation, PCR, and RT-PCR Total cellular RNA was extracted using RNAiso plus or All Prep DNA / RNA mini kit according to the product manual. RNA extraction was performed to synthesize cDNA using PrimeScript RT reagent Kit. cDNA was used to amplify specific genes by PCR or to quantify the expression levels of related genes by qPCR. qPCR reactions were prepared using SYBR Premix Ex Taq II (TaKaRa) and performed using AB VIIA7 RUO software (Applied Biosystems). Gapdh was used as a housekeeping gene.

[0076] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, unless they conflict with the explicit teachings of this specification. EXAMPLES

[0077] Below are examples illustrating procedures for carrying out the present invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

[0078] Example 1 - Amplification and construction of huCH101 To generate the humanized CH101 antibody and its variants, the variable heavy and variable light chains of mouse CH101 were amplified from selected hybridoma clones of CH101 using specific primer sets as described above. The cloned sequences were searched in the IMGT and V-BASE databases and aligned with the most compatible human germline selected for CDR grafting as shown in Figure 1 and Figure 2A-2B. There are a total of 112 amino acids in the variable heavy or kappa chain of human IgG1, 330 amino acids in the constant fragment, and 107 amino acids in the constant region of the kappa chain. When the variable region of CH101 was linked with the human IgG1 constant fragment, the constructed chimeric CH101 showed a humanization rate of 66.1% (437 / 661). Subsequently, the variable regions of the chimeric CH101 were further humanized, increasing the humanized portion to 97.0% (641 / 661) in huCH101 (or referred to as human framework hufrCH101) (FIG. 1).

[0079] Example 2 - Generation of humanized CH101 and its variants To exploit the therapeutic activity of CH101 variants, we made various modifications to the constant region of the chimeric or humanized version of CH101, resulting in a series of CH101 humanized antibodies, including CH101-huIgG1(N297A), CH101-huIgG1(L / S), CH101-huIgG4, huCH101-IgG1, huCH101-IgG4, and CH101-huIgG1 (chimeric CH101). The original CH101 (parent, fully mouse) was included as a positive control for these humanized versions. Because CH101 specifically binds to the conformational structure of Δ42PD1 expressed on the cell surface, 293T-Δ42PD1 and 293T-PD1 cell lines were established in the laboratory to evaluate the various versions of humanized CH101 antibodies by flow cytometry. All humanized antibodies could recognize Δ42PD1 expressed on the cell surface, but with different specificities (Figure 3A-3I). Detailed statistical analysis revealed the activity of each version of the humanized antibody in interacting with Δ42PD1 with different specificity and sensitivity (Table 1). As the data show, the parental mouse CH101 can detect an average of 59.20% of 293T-Δ42PD1 cells, while the sensitivity increases to 70.08% for the chimeric IgG1 version, but decreases to 45.46% for hufrCH101-IgG1. However, the cross-reactivity rate between CH101-huIgG1 and 293T-PD1 cells was on average 7.68%, compared to 3.15% for the parental mouse CH101. In contrast, almost no cross-reactivity was observed between huCH101-IgG1 and 293T-PD1 cells. Due to the limited expression of chimeric versions of CH101 using Fc engineering, including CH101-huIgG1(N297A), CH101-huIgG1(L / S), and CH101-huIgG4, the amount of these antibodies was insufficient for fluorescent labeling. When FACS staining was performed using a second anti-human Fc antibody, nonspecific staining was frequently observed, leading to nonspecific cross-reaction with 293T-PD1 cells. These antibodies therefore required further optimization for better production.

[0080] [Table 1]

[0081] Example 3 - Half-life of a humanized version of CH101 in NSG-PBL mice Next, three humanized CH101 antibodies, CH101-huIgG1, CH101-IgG4, and hufrCH101-IgG1, were evaluated in NSG-PBL mice. Each antibody was injected once at a dose of 200 μg per mouse, followed by analysis of mouse plasma samples obtained at different time intervals. The peripheral amounts of each antibody were then measured by ELISA to calculate detailed values ​​for each antibody's half-life. The plasma concentrations of all three investigated antibodies decreased over time within 72 hours after injection, followed by a plateau at a low level after 5 days. As shown in Figures 4A-4D, CH101-IgG4 had a half-life of 45.35 hr, which was slightly longer than the others compared to CH101-huIgG1 (44.55 hr) and hufrCH101-IgG1 (36.44 hr).

[0082] Example 4 - Chimeric CH101 as a therapeutic intervention tested in HCC mouse models Because the humanized NSG-huCD34 mouse model is not well established for HIV-1 antibody testing, we first evaluated the chimeric version of CH101, CH101-IgG1 (humanization rate 66.1%), for its potential as immunotherapy in the HCC mouse model. NSG-PBL mice were established as described previously. Huh7 cells were then subcutaneously inoculated (Figure 5A). Huh7 cells were grown in NSG-PBL mice from 1 week before antibody intervention. After antibody treatment, the time-course growth of Huh7 tumors was observed on a weekly basis until the endpoint (Figure 5B). At days 14 and 28 after tumor challenge (endpoint), there were significant differences between the antibody-treated and control groups, mainly in the CH101-huIgG1 and control groups (Figure 5C and Table 2). Specifically, the CH101-huIgG1 group was 3.83 mm larger on average than the hu-IgG1 control group on day 14. 3 At the same time, the parental mouse CH101 group showed smaller tumors (4.12 mm) compared to the mouse IgG1 control group. 3At endpoint, a significant reduction in tumor volume was consistently observed in the CH101-huIgG1 group, with a mean of 38.94 mm 3 However, there was no significant difference in tumor weight (Figure 5D) or in inflammatory cytokines released into plasma in 1 / 5 escaped mice (Figures 5E-5G).

[0083] [Table 2]

[0084] Example 5 - Treatment intervention study of hufrCH101-IgG1 in a mouse model of HCC We next evaluated hufrCH101-IgG1N, a 97.0% humanized version of CH101, for its hepatocellular carcinoma-suppressing effect in SG-PBL mice. Similarly, NSG-PBL mice were set up and challenged subcutaneously with Huh7 tumor cells (Figure 6A). Antibody treatment was administered daily for 5 days starting 1 week after tumor inoculation. Tumor growth in mice was measured and tumor volumes were recorded weekly until the humane endpoint (Figure 6B-6C). Encouragingly, tumor size was significantly reduced compared to the control group, mainly in the hufrCH101-IgG1 group (Figure 6C and Table 3). The hufrCH101-IgG1 group was 11.55 mm compared to the hu-IgG1 control group at day 14. 3 compared with 7.90 mm in the CH101-huIgG1 group. 3 Similarly, the average was 7.82 mm. 3 Tumor size progressed slowly in the two humanized CH101 antibody groups, reaching 13.33 mm for hufrCH101-IgG1 and 13.23 mm for hufrCH101-IgG1 at day 21, respectively. 3 , 14.56 mm for CH101-huIgG1 3 The control group had a score of 23.96 mm. 3 Thereafter, hufrCH101-IgG1 was 27.27 mm 3 Compared to the average of 14.26 mm 3The results showed relatively good control of tumor size at the end point (Table 3). No significant reduction in tumor weight (Figure 6D) or plasma inflammatory cytokine levels (Figures 6E-6G) was observed, which warrants further analysis.

[0085] Example 6 - Role of Δ42PD-1 in HCC tumorigenesis and resistance to ICB Immune checkpoint blockade (ICB) has improved cancer treatment, but the reason why many hepatocellular carcinoma (HCC) patients are resistant to PD-1 ICB remains unknown. Here, we revealed the role of Δ42PD-1, an isoform of programmed cell death protein 1 (PD-1), in HCC tumorigenesis and resistance to nivolumab ICB. Δ42PD-1 expression on different T cell subsets in HCC patients was detected as described in Materials and Methods above. Δ42PD-1 expression in PBMCs of 38 HCC patients and 43 healthy controls + T cells and PD-1 + T cells (Figures 7A-7B), Δ42PD-1 in PBMCs of these HCC patients + CD4 + T cells and CD8 + T cells (Figures 7C-7D), Δ42PD-1 in tumor and adjacent non-tumor tissues from 25 HCC patients + T cells or PD-1 + T cells (Figures 7E-7F), and in tumor and adjacent non-tumor tissues from the same patient. + CD4 + T cells or Δ42PD-1 + CD8 + The frequency of T cells (Figure 7G) was measured. In the same cohort of HCC patients, Δ42PD-1 + T cells and PD-1 + The correlation between the frequency of T cells and PD-1 in PBMCs and tumors of HCC patients stratified into UICC8 stage groups was evaluated (Figure 7H). + T cells and Δ42PD-1 + The frequency of T cells was also measured and is shown in Figure 7I.

[0086] We found a distinct T cell subset in untreated HCC patients that did not express PD-1 but expressed the isoform Δ42PD-1, accounting for up to 71% of cytotoxic T lymphocytes. + T cells infiltrated tumors and were positively correlated with the severity of HCC. + T cells express PD-1 + were more depleted than T cells.

[0087] Example 7 - Antitumor effect of anti-Δ42PD-1 antibody CH101 in mouse model HCC patients were investigated in three cohorts: untreated, nivolumab-treated, and isotype control-treated. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and tumor-infiltrating lymphocytes (TILs) were isolated from tumor tissues for immunophenotypic analysis. The functional significance of Δ42PD-1 was explored by single-cell RNA sequencing (scRNA-seq) analysis and verified by functional and mechanistic experiments. The effect of Δ42PD-1 monoclonal antibody was examined in an HCC humanized mouse model. The detailed experimental procedures are as described in Materials and Methods above.

[0088] Total human CD45 in the spleen or TME + CD3 + Among T cells, PD-1 + Δ42PD1 - T cells and PD-1 + Δ42PD-1 + Representative dot plots and frequencies of T cells were measured (Figure 8B-8C). Tumor growth curves of Huh7-NSG-HuPBL mice after CH101, anti-PD-1 nivolumab, or isotype treatment were shown in Figure 8E. Tumor growth curves of Huh7 in NSG mice, or Huh7-TLR4KO in NSG-HuPBL mice were shown in Figure 8F. Orthotopic MHCC-97L tumor growth was measured by bioluminescence imaging and shown in Figure 8H-8G. One representative liver image at the endpoint is shown (Figure 8I). Δ42PD-1 in HCC patients +Spearman correlations between T cell and IL-6 intensities were evaluated and are shown in Figure 8J. A representative image (40x) of a tumor sample from one patient shows that Δ42PD-1 + TIL cells and IL-6-producing TLR4 + The colocalization with HCC cells was shown (Figure 8K). Δ42PD-1 in PBMCs, spleens, and tumors of Huh7-NSG-huPBL mice at endpoint + T cells and PD-1 + Quantification of T cells is shown in Figure 8L. Quantification of plasma IL-6 and IL-8 at the endpoint is shown in Figure 8M. Representative immunofluorescence staining (20x magnification) of tumor tissues from Huh7-NSG-huPBL mice is shown with color coding of cellular expression of IL-6, TLR4, and CD3, respectively (Figure 8N).

[0089] The results showed that HCC patients treated with anti-PD-1 ICB showed effective PD-1 blockade, whereas Δ42PD-1 + The frequency of T cells increased over time, especially in patients with progressive disease. + T cells likely participated in tumorigenesis and maintained HCC through TLR4 signaling. The anti-Δ42PD-1 antibody CH101, but not nivolumab, suppressed tumor growth in a mouse HCC model.

[0090] This finding not only revealed the mechanism underlying resistance to PD-1 ICB but also identified the anti-Δ42PD-1 antibody CH101 for immunotherapy of HCC.

[0091] Example 8 - Amplification and construction of huCH34 To generate the humanized CH34 antibody and its variants, the variable heavy and light chains of mouse CH34 were amplified from selected hybridoma clones of CH34 using methods similar to those described in Example 1.

[0092] The amino acid sequences of the heavy and light chains of the murine CH34 mAb are shown in Figure 9. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the humanized CH34 mAb are shown in Figure 10.

[0093] Example 9 - Specificity of anti-Δ42PD1 antibody CH34 binding to Δ42PD-1 expressed on human 293T cells To examine the specificity of anti-Δ42PD1 antibody CH34 and anti-PD1 antibody (EH12.2H7), 293T cells were transfected with Δ42PD-1, plasmid PCDH expressing PD-1, or vector alone. Untransfected cells were included as controls. Two days after transfection, the specificity of anti-Δ42PD1 antibody CH34 and anti-PD1 antibody EH12.2H7 is measured by analysis of PD-1 and Δ42PD1 expression.

[0094] The results are shown in Figure 11. The results show that the anti-Δ42PD1 antibody CH34 specifically binds to Δ42PD1, but does not bind to PD-1 expressed in human 293T cells.

[0095] Example 10 - Specificity of anti-Δ42PD1 antibody CH34 for Δ42PD-1 expressed on human B cells To test the specificity of anti-Δ42PD1 antibody CH34 compared to anti-PD1 antibody (EH12.2H7), RPMI8866 B cells were transfected with plasmid PCDH expressing Δ42PD-1-GFP, PD-1-GFP, or GFP by the Neon electroporation system. Untransfected RPMI8866 B cells were included as controls. Two days after transfection, transfection efficiency was determined by flow cytometric analysis of GFP (upper panel). The specificity of anti-Δ42PD1 antibody CH34 and anti-PD1 antibody EH12.2H7 was measured by analyzing the expression of PD-1 and Δ42PD1 (lower panel).

[0096] The results are shown in Figure 12. The results show that the anti-Δ42PD1 antibody CH34 specifically binds to Δ42PD1, but does not bind to PD-1 expressed on human B cells.

[0097] Example 11 - Increased Δ42PD-1 expression with B cell depletion in CHP The molecular mechanisms that control BCR-mediated human B cell activation and fate remain incompletely understood. Also, HIV-1 antibody responses drain into exhausted B cells, but the inhibitors of B cell activation following B cell receptor (BCR) stimulation remain incompletely understood. To explore these mechanisms, we measured the expression of Δ42PD-1 and B cell exhaustion in BD and chronic HIV-1 patients (CHP). CD19 from BD and untreated CHP were expressed in Δ42PD-1 and B cell exhaustion. + Representative flow cytometry analysis of B cells is shown in Figure 13A. Figure 13B summarizes the data in Figure 13A and shows the fluorescence intensity (MFI) and Δ42PD-1 in BD and CHP. + The frequency of B cells is shown. - CD27 + Activated memory (AM), CD21 - CD27 + Static Memory (RM), CD27 - CD21 - Tissue-like memory and anergy naive B (TLM / aN), and CD27 - CD21 + To identify resting naive plus intermediate memory B cells (IM / rN), gating was performed on CD21 / CD27 stained B cells from representative BDs and CHPs, and the results are shown in Figure 13C. Figure 13D summarizes the data from Figure 13C, showing increased frequencies of AM and TLM / aN B cells in CHPs compared to BDs. Expression of Δ42PD-1 in representative BD and CHP AM, TLM / aN, RM, and IM / rN B cells is shown in Figure 13E. Figure 13F summarizes the data from Figure 13E, showing the MFI of Δ42PD-1 in B cell subsets from BDs and CHPs. PD-1 + and Δ42PD-1+ Representative flow cytometry analyses of AM, TLM / aN, RM, and IM / rN B cells from the BD and CHP of each patient are shown in Figure 13G. Figure 13H summarizes the data in Figure 13G and shows that Δ42PD-1 in the BD and CHP + The frequencies of AM, TLM / aN, RM, and IM / rN B cells are shown.

[0098] These results indicate that Δ42PD-1 is significantly more highly expressed in activated memory B cells, anergic naive B cells, and tissue-like memory B cells than CHP PD-1 and FcRL4.

[0099] Example 12 - Anti-Δ42PD-1 antibodies decrease recruitment of SHP1 and promote the AKT1 / FOXO1 pathway and B cell proliferation To examine whether CH34 promotes B cell proliferation by BCR stimulation, flow cytometry was performed. Primary B cells (n=6) from BD were prelabeled with FITC-CFSE and stimulated with α-IgM / GF(ab')2 (10 μg / ml) and msIgG2b control antibody, or CH34. Cells were analyzed by flow cytometry on day 5. The results are shown in Figures 14A-14B.

[0100] To determine whether CH34 reduces the recruitment of SHP1 by Δ42PD-1, primary B cells from BD were stimulated with α-IgM / GF(ab')2 (10 μg / ml) and msIgG2b control antibody or CH34, lysed on day 2, and subjected to immunoprecipitation and Western blotting with antibodies against Δ42PD-1 and SHP1, respectively. The results are shown in Figure 14C.

[0101] Ki67 +To examine the effect of CH34 treatment on B cell proliferation, phoph-AKT1-S473 and phosph-FOXO1-S256 levels, and P27 protein expression, as well as FOXO1 and P27 mRNA expression in B cells, primary B cells were cultured in complete RPMI1640 medium supplemented with α-IgM / GF(ab')2 (10 μg / ml) and msIgG2b control antibody (10 μg / ml) or CH34 (10 μg / ml), or SHP1 inhibitor NSC87877 (10 uM). After 48 hours, cells were stained and subjected to flow cytometry analysis or lysed and subjected to Western blotting detection of AKT1, FOXO1, and P27, or qPCR of FOXO1 and P27 mRNA. The results are shown in Figure 14D-14F.

[0102] The results showed that CH34 promoted B cell proliferation by BCR stimulation, CH34 reduced Δ42PD-1-induced SHP1 recruitment, and CH34 treatment downregulated Ki67. + We expand the B cell population and show that CH34 treatment increases phoph-AKT1-S473 and phosph-FOXO1-S256 levels and decreases P27 protein expression, and that CH34 treatment increases FOXO1 and P27 mRNA in B cells.

[0103] Example 13 - CH34 promotes B cell proliferation following BCR stimulation in HIV patients To investigate the effect of CH34 on B cell proliferation after BCR stimulation in HIV patients, we performed flow cytometry. IgD isolated from PBMCs of CHP + B cells and IgD -B cells were prelabeled with CFSE and cultured in RPMI 1640 medium supplemented with α-IgM / GF(ab')2 (10 μg / ml), TD or TI stimulation + CH34 (10 μg / ml), or msIgG2b control (10 μg / ml) for 5 days and analyzed by flow cytometry. Statistical significance was determined using a general one-way ANOVA and the Benjamini, Krieger and Yekutieli two-step linear step-up procedure with single pool variance. Results are shown in Figures 15A-15H.

[0104] The results showed that CH34 inhibited the expression of naive B cells (IgD + ) and memory B cells (IgD - ) (Figures 15A-15B), increases CD86 expression in naive and memory B cells derived from the CHP by BCR stimulation (Figures 15C-15D), increases the proliferative response of B cells from the CHP after TD stimulation (Figures 15E-15F), and increases the proliferative response of naive B cells derived from the CHP after TI stimulation.

[0105] The results suggest that BCR stimulation upregulates Δ42PD-1, resulting in B cell exhaustion, cell cycle arrest, and death. Mechanistically, Δ42PD-1 recruits Src homology region 2 domain-containing phosphatase 1 (SHP1) via an intracellular immunoreceptor tyrosine-based switch motif (ITSM). SHP1 then binds to AKT1 and inhibits AKT1 activation, thereby suppressing the AKT1 / FOXO1 pathway. However, the Δ42PD-1-specific antibody CH34 reduces SHP1 recruitment and increases AKT1 / FOXO1 activation and proliferation of CHP-derived B cells (Figure 16).

[0106] These findings indicate that Δ42PD-1 is a previously unrecognized suppressor of BCR-mediated B-cell activation and may be a potential immunotherapeutic target to restore B-cell function.

[0107] array SEQ ID NO:1 protein Artificial sequence: CH101 VH amino acid sequence EVQLEESGPDLVKPSQSLSLTCTVTGYSITSGFAWHWLRQFPGNRLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATFYCARSDFWGQGTFVTVSS

[0108] SEQ ID NO:2 protein Artificial sequence: CH101 VK amino acid sequence DVVMTQTPLNLSVTFGQPVSISCTSSQSLLDRDGETFMNWLFQRPGQSPKRLIYLVSKLDFGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPLTFGGGTKLEIKRGQAG

[0109] SEQ ID NO:3 protein Artificial sequence: hufrCH101 VH amino acid sequence QVQLQESGPGLVKPSETLSLTTCTVSGYSITSGFAWHWIRQPPGKGLEWIGYIHYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSDFWGQGTLVTVSS

[0110] SEQ ID NO:4 protein Artificial sequence: hufrCH101 VK amino acid sequence DVVMTQSPLSLPVTLGQPASISCTSSQSLLDRDGETFMNWFQQRPGQSPRRLIYLVSKLDFGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCWQGTHFPLTFGGGTKVEIKRGQAG

[0111] SEQ ID NO:5 DNA Artificial sequence: CH101 VH nucleic acid sequence GAGGTGCAGCTCGAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTATTCCATCACCAGTGGTTTTGCCTGGCACTGGCTCCGGCAGTTTCCAGGAAACAGACTGGAATGGATGGGCTACATACACTATAGTGGA AGCACTAACTACAACCCATCTCTCAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAATCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACTTTTTACTGTGCAAGGTCTGACTTCTGGGGCCAAGGCACCTTTGTCACAGTCTCCTCA

[0112] sequence number 6 DNA Artificial sequence: CH101 VK nucleic acid sequence GATGTTGTGATCCAGCAGACTCCACTCAATTTGTCGGTCACCTTTGGACAACCAGTCTCCATCTCTTGCACGTCTAGTCAGAGCCTCTTAGATCGTGATGGAGAGACATTCATGAATTGGTTGTTCCAGCGGCCAGGCCAGTTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAC TGGACTTTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTGCTGGCAAGGTACACATTTTCCTCTCACGTTCGGTGGTGGGACCAAGCTGGAAATAAAACGCGGCCAGGCCGGC

[0113] sequence number 7 DNA Artificial sequence:hufrCH101 VH nucleic acid sequence CAGGTCAGCTCCAGGAGTCAGGACCCGGTCTGGTGAAACCTTCAGAGACACTGAGTCTGACATGTACGGTAAGTGGGTACTCCATTACTTCCGGTTTTGCTTGGCATTGGATACGGCAACCGCCTGGGAAGGGGTTGGAGTGGATAGGTTATATTCATTATAGCGGG TCAACAAATTATAACCCATCACTCAAGTCCCGCGTCACGATAAGCGTTGATACCTCCAAGAATCAGTTCAGCTTGAAATTGTCAAGCGTAACAGCCGCCGATACCGCAGTGTATTATTGCGCAAGGAGTGACTTTTGGGGCCAAGGTACGCTTGTCACCGTAAGTTCC

[0114] SEQ ID NO:8 DNA Artificial sequence: hufrCH101 VK nucleic acid sequence GACGTAGTCATGACCCAGTCCCCACTGTCATTGCCGGTAACGCTTGGACAGCCTGCTAGTATCTCATGCACATCCAGCCAATCCCTCCTTGACCGCGACGGTGAGACGTTTATGAATTGGTTCCAGCAGAGACCAGGACAGTCACCGAGGAGACTTATTTACCTTGTTTCAAAGC TCGACTTCGGTGTCCCCGACAGATTTAGCGGTAGTGGTTCTGGGACAGATTTCACCCTTAAAATCAGTAGGGTCGAAGCGGAGGACGTGGGTGTCTACTATTGTTGGCAAGGCACGCACTTTCCATTGACCTTTGGCGGGGGGACAAAGGTGGAAATTAAACGGGCCAGGCGGGG

[0115] SEQ ID NO:9 protein Homo sapiens: Amino acid sequence of germline variable heavy chain with respect to CDR grafting (GenBank: BAA75026.1, Accession number: AB019437.1) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

[0116] SEQ ID NO:10 protein Homo sapiens: Amino acid sequence of germline variable kappa chain with respect to CDR grafting (GeneBank: BAC01687.1, Accession number: AB064059.1) DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPP

[0117] SEQ ID NO:11 protein Homo sapiens: Amino acids in the J region of human heavy chains (IGHJ4*01) YFDYWGQGTLVTVSS

[0118] SEQ ID NO:12 protein Homo sapiens: Amino acids in the J region of the human kappa chain (IGKJ4*01) TFGGGTKVEIK

[0119] SEQ ID NO:13 DNA Homo sapiens: Germline variable heavy chain nucleic acid sequence for CDR grafting (GenBank: BAA75026.1, Accession number: AB019437.1) CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGGCGGCAGCGTGAGCAGCGGCGGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGGGCCTGGAGTGGA TCGGCTACATCTACTACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGG

[0120] SEQ ID NO:14 DNA Homo sapiens: Germline variable kappa chain nucleic acid sequence for CDR grafting (GeneBank: BAC01687.1, Accession number: AB064059.1) GACGTGGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGTACAGCGACGGCAACACCTACCTGAACTGGTTCCAGCAGAGGCCCGGCCAGAGCCCCAGGA GGCTGATCTACAAGGTGAGCAACAGGGACAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGGCACCCACTGGCCCCCC

[0121] SEQ ID NO:15 DNA Homo sapiens: Nucleic acid sequence of the J region of the human heavy chain (IGHJ4*01) TACTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA

[0122] SEQ ID NO:16 DNA Homo sapiens: Nucleic acid sequence of the J region of the human kappa chain (IGKJ4*01) ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0123] SEQ ID NO:17 DNA Homo sapiens: Human kappa chain constant fragment nucleic acid sequence CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0124] SEQ ID NO:18 protein: Homo sapiens: Human kappa chain constant fragment nucleic acid sequence amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0125] SEQ ID NO:19 protein Homo sapiens: Human heavy chain constant fragment amino acid sequence (IgG1) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0126] SEQ ID NO:20 DNA Homo sapiens: Human heavy chain constant fragment nucleic acid sequence (IgG1) GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA

[0127] SEQ ID NO:21 protein Homo sapiens: Human heavy chain constant fragment amino acid sequence (IgG4) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0128] SEQ ID NO:22 DNA Homo sapiens: Human heavy chain constant fragment nucleic acid sequence (IgG4) GCGTCGACCAAGGGACCAAGCGTATTCCCGCTTGCGCCATGTAGCAGATCAACAAGTGAGTCCACGGCGGCCCTTGGGTGCTTGGTAAAGGATTACTTTCCTGAGCCAGTAACCGTGTCTTGGAATTCAGGAGCTCTCACGTCAGGGGTACACACCTTCCCTGCCGTCCTTCAATCTTCTGGCCTCTATAGCCTCTCAAGCGTCGTTACTGTGCCATCCTCCTCACTCGGAACTAAAACATATACTTGCAACGTAGACCACAAGCCCTCAAATACAAAAGTTGACAAACGAGTTGAATCTAAGTACGGCCCGCCATGTCCGCCATGTCCAGCCCCCGAATTCTTGGGGGGACCATCCGTATTTCTTTTCCCGCCGAAACCCAAAGACACCTTGATGATAAGCCGAACACCAGAGGTTACATGTGTCGTTGTTGATGTCTCTCAGGAAGATCCTGAGGTCCAGTTTAACTGGTACGTAGATGGGGTGGAAGTTCACAACGCAAAAACCAAGCCGAGAGAGGAACAGTTCAACTCAACCTATCGGGTTGTTTCCGTTCTCACAGTTCTGCATCAGGACTGGCTTAATGGAAAAGAATACAAGTGTAAAGTGTCAAATAAGGGCCTCCCATCAAGCATTGAAAAGACCATATCTAAGGCCAAAGGCCAACCTCGCGAGCCTCAAGTCTACACCTTGCCTCCTAGCCAAGAAGAAATGACAAAGAACCAAGTATCTCTCACATGTCTTGTTAAAGGGTTCTATCCAAGCGATATCGCTGTTGAGTGGGAAAGCAATGGCCAACCTGAAAATAACTACAAAACCACTCCTCCCGTGCTTGACTCAGATGGTTCTTTCTTCCTCTATTCACGATTGACTGTGGACAAAAGTCGCTGGCAGGAAGGGAACGTTTTCTCCTGTAGCGTGATGCACGAGGCGCTCCACAATCATTATACCCAAAAGTCACTCTCTCTTAGCCTTGGCAAG

[0129] SEQ ID NO:23 protein Artificial sequence: CH101 VH CDR1 (Kabat numbering system) GYSITSGF

[0130] SEQ ID NO:24 protein Artificial sequence: CH101 VH CDR2 (Kabat numbering system) HYSGS

[0131] SEQ ID NO:25 protein Artificial sequence: CH101 VH CDR3 (Kabat numbering system) SDF

[0132] SEQ ID NO:26 protein Artificial sequence: CH101 VL CDR1 (Kabat numbering system) TSSQSLLDRDGETFMN

[0133] SEQ ID NO:27 protein Artificial sequence: CH101 VL CDR2 (Kabat numbering system) LVSKLDF

[0134] SEQ ID NO:28 protein Artificial sequence: CH101 VH CDR3 (Kabat numbering system) WQGTHFPL

[0135] SEQ ID NO:29 protein Artificial sequence: CH34 VH CDR1 (Kabat numbering system) GFTFSDYG

[0136] SEQ ID NO:30 protein Artificial sequence: CH34 VH CDR2 (Kabat numbering system) ISLAMYTI

[0137] SEQ ID NO:31 protein Artificial sequence: CH34 VH CDR3 (Kabat numbering system) ARGGAGFAY

[0138] SEQ ID NO:32 protein Artificial sequence: CH34 VL CDR1 (Kabat numbering system) DHINNW

[0139] SEQ ID NO:33 protein Artificial sequence: CH34 VL CDR2 (Kabat numbering system) GAT

[0140] SEQ ID NO:34 protein Artificial sequence: CH34 VL CDR3 (Kabat numbering system) QQYWSTPWT

[0141] SEQ ID NO:35 protein Artificial sequence: Mouse CH34 VH EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAPGKGPEWVAFISSLAYTIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARGGAGFAYWGQGTLVTVS

[0142] SEQ ID NO:36 protein Artificial sequence: Mouse CH34 VL DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK

[0143] SEQ ID NO:37 protein Artificial sequence: humanized CH34 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMSWIRQAPGKGLEWVSYISSLAYTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGAGFAYWGQGTLVTVS

[0144] SEQ ID NO:38 protein Artificial sequence: humanized CH34 VL DIQMTQSPSSLSASVGDRVTITCRASDHINNWLAWYQQKPGKAPKLLLYGATRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYWSTPWTFGGGTKVEIK

[0145] SEQ ID NO:39 protein Artificial sequence: signal peptide MGWSCIILFLVATATGVHS

[0146] SEQ ID NO:40 protein Artificial sequence: Mouse CH34 heavy chain EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAPGKGPEWVAFISSLAYTIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARGGAGFAYWGQGTLVT VSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINCPP PCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIER TISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0147] SEQ ID NO:41 protein Artificial sequence: Mouse CH34 kappa chain DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0148] It is to be understood that the examples and embodiments described herein are for illustrative purposes, and that various modifications or changes in light of them will be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims. In addition, any element or limitation of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) disclosed herein, or with any other invention or embodiment thereof, and all such combinations are contemplated within the scope of the present invention, without limitation thereto.

Claims

1. Humanized Δ42PD1-specific monoclonal antibody (mAb).

2. the variable heavy chain comprises at least 80% sequence identity to SEQ ID NO:3, and the variable light chain comprises at least 80% sequence identity to SEQ ID NO:4; 2. The humanized Δ42PD1-specific mAb of claim 1.

3. The heavy and light chain constant regions are A human IgG1 constant heavy chain fragment, which is SEQ ID NO: 19 or a sequence having at least 80% identity to SEQ ID NO: 19, and 2. The humanized Δ42PD1-specific mAb of claim 1, which is a human IgG1 light chain fragment having a sequence of SEQ ID NO:18 or having at least 80% identity to SEQ ID NO:

18.

4. A composition comprising the humanized Δ42PD1-specific mAb of claim 1.

5. The composition of claim 4 further comprising a pharma- ceutically acceptable carrier and / or excipient.

6. A nucleic acid molecule encoding a humanized Δ42PD1-specific mAb.

7. The nucleic acid molecule of claim 6 which is a plasmid.

8. The nucleic acid sequence encoding the variable heavy chain of the humanized Δ42PD1 specific mAb comprises at least 80% sequence identity with SEQ ID NO:7, and 7. The nucleic acid molecule of claim 6, wherein the nucleic acid sequence encoding the variable light chain of the humanized Δ42PD1-specific mAb comprises at least 80% sequence identity with SEQ ID NO:

8.

9. The nucleic acid sequences encoding the heavy and light chain constant regions are A human IgG1 constant heavy chain fragment, which is SEQ ID NO: 20 or a sequence having at least 80% sequence identity with SEQ ID NO: 20; and a human constant light chain fragment, which is SEQ ID NO: 17 or a sequence having at least 80% sequence identity with SEQ ID NO: 17; The nucleic acid molecule of claim 6, which is a nucleic acid sequence encoding

10. A pharmaceutical composition for inhibiting cancer cell proliferation in a subject, comprising a humanized Δ42PD1-specific monoclonal antibody (mAb).

11. The variable heavy chain of the humanized Δ42PD1 specific mAb comprises at least 80% sequence identity with SEQ ID NO:3, and 11. The pharmaceutical composition of claim 10, wherein the variable light chain of the humanized Δ42PD1-specific mAb comprises at least 80% sequence identity with SEQ ID NO:

4.

12. 11. The pharmaceutical composition of claim 10, wherein the heavy and light chain constant regions of the humanized Δ42PD1-specific monoclonal antibody are human IgG1 constant heavy chain fragments and constant light chain fragments.

13. the human IgG1 constant heavy chain has the sequence of SEQ ID NO: 19 or a sequence having at least 80% identity with SEQ ID NO: 19; and The human IgG1 light chain fragment has the sequence of SEQ ID NO: 18 or a sequence having at least 80% identity with SEQ ID NO: 18; The pharmaceutical composition according to claim 12.

14. 1. A Δ42PD1 specific antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), (i) the VH comprises HCDR1-3 having the amino acid sequences set forth in SEQ ID NOs:23-25, respectively, and the VL comprises HCDR1-3 having the amino acid sequences set forth in SEQ ID NOs:26-28, respectively; or (ii) VH comprises HCDR1-3 having the amino acid sequences shown in SEQ ID NOs: 29-31, respectively, and VL comprises LCDR1-3 having the amino acid sequences shown in SEQ ID NOs: 32-34, respectively; Δ42PD1-specific antibodies or antigen-binding fragments thereof.

15. 15. The antibody or antigen-binding fragment thereof of claim 14, (i) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2; or (ii) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4; or (iii) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:35, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:36; or (iv) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; An antibody or an antigen-binding fragment thereof.

16. 15. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody is an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

17. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

18. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody comprises a heavy chain constant region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19 or 21, and / or a light chain constant region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

18.

19. The antigen-binding fragment may be Fab, Fab', F(ab') ２15. The antibody or antigen-binding fragment thereof of claim 14, selected from the group consisting of Fd, Fd', Fv, scFv, ds-scFv, and dAb.

20. 15. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody is a murine, human, or humanized antibody.

21. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody is a monoclonal antibody.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the antibody comprises a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

41.

23. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody is a bispecific or multispecific antibody.

24. 24. The antibody or antigen-binding fragment thereof of claim 23, wherein the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen.

25. 25. The antibody or antigen-binding fragment thereof of claim 24, wherein the second antigen is a tumor-associated antigen or an immune cell antigen, such as a T cell antigen.

26. 26. A nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to any one of claims 14 to 25.

27. A vector comprising the nucleic acid of claim 26.

28. 27. A host cell comprising the nucleic acid of claim 26.

29. 26. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 14 to 25.

30. (i) an antibody or antigen-binding fragment thereof according to any one of claims 14 to 25, or an antibody-drug conjugate according to claim 29; (ii) a pharma- ceutically acceptable carrier or adjuvant.

31. 31. The pharmaceutical composition of claim 30 for use in a method of treating a disease in a subject, wherein the disease is cancer or a disease or condition caused by a viral infection.

32. 32. The pharmaceutical composition of claim 31, wherein the disease is hepatocellular carcinoma or a disease or condition caused by HIV infection.

33. 32. The pharmaceutical composition of claim 31, wherein the method further comprises administering a second therapeutic agent to the subject.

34. 34. The pharmaceutical composition of claim 33, wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

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