CD8-specific binding protein and its method of use

Abstract

The disclosure provides binding agents that specifically bind to CD8 (e.g., antibodies or their antigen-binding fragments, binding proteins), and their use in the treatment of diseases or disorders such as inflammatory diseases or autoimmune diseases. In some embodiments, the disclosure provides binding domains and binding proteins that target CD8a. In some embodiments, the disclosure provides binding proteins comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 133 (wherein X=G or C), and (b) a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 144 (wherein (1) X1=I or F, (2) X2=G or C, (3) X3=F or V, (4) X4=A or F, (5) X5=A or R, and X6=G or A).

Description

[Technical Field]

[0001] Reference to electronic sequence listings This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on 20 May 2024, is named SeqList-368576-41002.xml and has a size of 166,696 bytes. [Background technology]

[0002] background CD8 is a protein expressed on the surface of many different types of immune cells, including approximately 90% of cytotoxic T lymphocytes (Cole et al., Imunology 137:139-148, 2012). It functions as a co-receptor during antigen binding by the T cell receptor (TCR), stabilizing the interaction between the TCR and the peptide major histocompatibility complex (pMHC) molecule and providing a co-activation signal (Cole et al., 2012, see above). Antibodies and similar binding proteins that target CD8 can be used as immune system modulators. For example, activating anti-CD8 antibodies can be used to induce effector function in CD8+ T cells (Clement et al., J Immunol 187(2):654-663, 2011), while blocking anti-CD8 antibodies can be used to suppress autoreactive CD8+ T cells (Clement et al., Scientific Reports Sci Rep 6:35332, 2016). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Cole et al., Imunology 137:139-148, 2012. [Non-Patent Document 2] Clement et al., J Immunol 187(2):654-663, 2011. [Non-Patent Document 3] Clement et al., Scientific Reports Sci Rep 6:35332, 2016 Summary of the Invention Means for Solving the Problems

[0004] Brief Summary In some embodiments, the present disclosure provides a binding domain and a binding protein that target CD8a.

[0005] In some embodiments, the present disclosure

Chemical formula

[0006] In some embodiments, the present disclosure

Chemical formula

[0007] In some embodiments, the present disclosure provides a binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence according to SEQ ID NO: 133 (where X = G or C), and (b) a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence according to SEQ ID NO: 144 (where (1) X1 = I or F, (2) X2 = G or C, (3) X3 = F or V, (4) X4 = A or F, (5) X5 = A or R, and X6 = G or A).

[0008] In some embodiments, the present disclosure provides a binding protein comprising one or more of: (a) CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (b) the following framework regions: (i) light chain FR4 according to SEQ ID NO: 126; (ii) heavy chain FR2 according to SEQ ID NO: 128; (iii) heavy chain FR3 according to SEQ ID NO: 129; (iv) heavy chain FR3 according to SEQ ID NO: 130; (v) heavy chain FR3 according to SEQ ID NO: 131; (vi) heavy chain FR3 according to SEQ ID NO: 132; and (vii) heavy chain FR3 according to SEQ ID NO: 16 having one or more of the following substitutions at the amino acid positions within SEQ ID NO: 16: F6V, A10F, A14R, and G39A.

[0009] In some embodiments, the present disclosure provides a binding protein comprising one or more of: (a) CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO: 127, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (b) the following framework regions: (i) light chain FR4 according to SEQ ID NO: 126; (ii) heavy chain FR2 according to SEQ ID NO: 128; (iii) heavy chain FR3 according to SEQ ID NO: 129; (iv) heavy chain FR3 according to SEQ ID NO: 130; (v) heavy chain FR3 according to SEQ ID NO: 131; (vi) heavy chain FR3 according to SEQ ID NO: 132; (vii) heavy chain FR3 according to SEQ ID NO: 16 having one or more of the following substitutions at the amino acid positions within SEQ ID NO: 16: F6V, A10F, A14R, and G39A; (viii) light chain framework region of VL according to SEQ ID NO: 7; and (ix) heavy chain framework region of VH according to SEQ ID NO: 8.

[0010] In some embodiments, the present disclosure provides a binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 55 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 56, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 55 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 56, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 55 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 56.

[0011] In some embodiments, pharmaceutical compositions comprising a binding domain or binding protein disclosed herein are provided.

[0012] In some further embodiments, the Disclosure provides methods for using the binding proteins or pharmaceutical compositions disclosed herein, for example, methods for activating CD8+ regulatory T cells (CD8+ Treg cells), and methods for treating or preventing diseases (e.g., autoimmune diseases). [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows the "bottle opener" structure of the bispecific binding protein.

[0014] [Figure 2] Figure 2 shows the binding of anti-KIR2DL / anti-CD8a parent and variant 20 to CD8 SKW cells.

[0015] [Figure 3] Figure 3 shows the binding of anti-KIR2DL / anti-CD8a parent and variant 20 to primary CD8 T cells.

[0016] [Figure 4]Figure 4 shows the single-dose PK / tolerance study design in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. Subsequently, mice exhibiting over 25% hCD45, over 3% hCD3, and over 2% hCD56 were sent to the laboratory.

[0017] [Figure 5A-B] Figures 5A–5D show the binding of variant 20 to KIR+CD8 cells and NK cells, as well as the associated activation levels of KIR+CD8 cells and the exclusion of CD4+ cells. KIR+CD8 in all PBMCs was selectively gated using a non-blocking anti-KIR antibody, and NK cells were defined using an anti-NKp46 antibody. Binding to different cell populations was detected using an anti-human IgG1 Fc secondary antibody. Intracellular granzyme B was detected using an anti-granzyme B antibody after intracellular staining. Figure 5A shows the binding of variant 20 to KIR+CD8 cells in PBMCs derived from a celiac disease donor. Figure 5B shows the reduction of KIR+CD8 cells in celiac disease donor PBMCs incubated with variant 20 compared to cells incubated with a control monospecific antibody. Figure 5C shows the binding of variant 20 to NK cells in PBMCs derived from a normal donor. Figure 5D shows the elimination of gliadin-responsive celiac disease donor CD4+ T cells, detected by an increase in annexin V+CD4+ T cells 48 hours after the addition of variant 20. In Figures 5A-5C, circles, inverted triangles, and rhombuses represent the results for variant 20, CD8 monospecific antibody, and KIR2DL monospecific antibody, respectively. [Figure 5C-D]Figures 5A–5D show the binding of variant 20 to KIR+CD8 cells and NK cells, as well as the associated activation levels of KIR+CD8 cells and the exclusion of CD4+ cells. KIR+CD8 in all PBMCs was selectively gated using a non-blocking anti-KIR antibody, and NK cells were defined using an anti-NKp46 antibody. Binding to different cell populations was detected using an anti-human IgG1 Fc secondary antibody. Intracellular granzyme B was detected using an anti-granzyme B antibody after intracellular staining. Figure 5A shows the binding of variant 20 to KIR+CD8 cells in PBMCs derived from a celiac disease donor. Figure 5B shows the reduction of KIR+CD8 cells in celiac disease donor PBMCs incubated with variant 20 compared to cells incubated with a control monospecific antibody. Figure 5C shows the binding of variant 20 to NK cells in PBMCs derived from a normal donor. Figure 5D shows the elimination of gliadin-responsive celiac disease donor CD4+ T cells, detected by an increase in annexin V+CD4+ T cells 48 hours after the addition of variant 20. In Figures 5A-5C, circles, inverted triangles, and rhombuses represent the results for variant 20, CD8 monospecific antibody, and KIR2DL monospecific antibody, respectively.

[0018] [Figure 5E]Figures 5E–5F show cytokine release in primary PBMCs derived from 10 healthy human donors in a wet-coated presentation format. Variant 20 ("Var20") at various concentrations (0.032 μg / mL, 0.16 μg / mL, 0.8 μg / mL, 4 μg / mL, 20 μg / mL, and 100 μg / mL) were evaluated in the assay. Treatment with anti-CD3 antibody (OKT3 clone; positive control), human IgG1 antibody (negative control), and a "no treatment" control were also included in the assay. All treatments were evaluated in triplicates in both soluble and wet-coated plate formats. Tissue culture supernatants from treated PBMC samples were collected 24 hours after treatment. Levels of IL-2, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were measured using an MSD platform. All donors were responsive to positive control anti-CD3 (Figure 5E: IL-6, Figure 5F: TNF-α), demonstrating that these donors possess the ability to release cytokines in response to immunomodulatory stimuli. In both treatment modes, there was no dose-responsive cytokine release (IL-2, IL-6, IL-8, IL-10, and TNF-α) stimulated by variant 20 above isotype or untreated control levels for any donor (e.g., Figure 5E for IL-6, Figure 5F for TNF-α). [Figure 5F]Figures 5E–5F show cytokine release in primary PBMCs derived from 10 healthy human donors in a wet-coated presentation format. Variant 20 ("Var20") at various concentrations (0.032 μg / mL, 0.16 μg / mL, 0.8 μg / mL, 4 μg / mL, 20 μg / mL, and 100 μg / mL) were evaluated in the assay. Treatment with anti-CD3 antibody (OKT3 clone; positive control), human IgG1 antibody (negative control), and a "no treatment" control were also included in the assay. All treatments were evaluated in triplicates in both soluble and wet-coated plate formats. Tissue culture supernatants from treated PBMC samples were collected 24 hours after treatment. Levels of IL-2, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were measured using an MSD platform. All donors were responsive to positive control anti-CD3 (Figure 5E: IL-6, Figure 5F: TNF-α), demonstrating that these donors possess the ability to release cytokines in response to immunomodulatory stimuli. In both treatment modes, there was no dose-responsive cytokine release (IL-2, IL-6, IL-8, IL-10, and TNF-α) stimulated by variant 20 above isotype or untreated control levels for any donor (e.g., Figure 5E for IL-6, Figure 5F for TNF-α).

[0019] [Figure 6A]Figures 6A–6F show the frequency of human immune cells in the peripheral blood of huCD34+NSG-Tg(Hu-IL15) mice after a single dose of variant 20. The frequency of immune cell subsets, including CD4+ or CD8+ T cell subsets (Figures 6A–6D) and NK cells (Figures 6E–6F), was assessed by flow cytometry in peripheral blood at baseline, day 14, and day 28 after IV administration of variant 20. At baseline, registered study mice had a mean of 62% hCD45, 5% CD3, and 7% CD56. Figure 6A shows the frequency (cells / μL) of KIR+ hCD8 T cells. Figure 6B shows the frequency (cells / μL) of hCD4 T cells. Figure 6C shows the frequency (cells / μL) of hCD8 T cells. Figure 6D shows the ratio of CD4 T cells to CD8 T cells. Figure 6E shows the frequency of hCD56+ NK cells (cells / μL). Figure 6F shows the frequency of KIR+ hCD56+ NK cells (cells / μL). [Figure 6B] Figures 6A–6F show the frequency of human immune cells in the peripheral blood of huCD34+NSG-Tg(Hu-IL15) mice after a single dose of variant 20. The frequency of immune cell subsets, including CD4+ or CD8+ T cell subsets (Figures 6A–6D) and NK cells (Figures 6E–6F), was assessed by flow cytometry in peripheral blood at baseline, day 14, and day 28 after IV administration of variant 20. At baseline, registered study mice had a mean of 62% hCD45, 5% CD3, and 7% CD56. Figure 6A shows the frequency (cells / μL) of KIR+ hCD8 T cells. Figure 6B shows the frequency (cells / μL) of hCD4 T cells. Figure 6C shows the frequency (cells / μL) of hCD8 T cells. Figure 6D shows the ratio of CD4 T cells to CD8 T cells. Figure 6E shows the frequency of hCD56+ NK cells (cells / μL). Figure 6F shows the frequency of KIR+ hCD56+ NK cells (cells / μL). [Figure 6C]Figures 6A–6F show the frequency of human immune cells in the peripheral blood of huCD34+NSG-Tg(Hu-IL15) mice after a single dose of variant 20. The frequency of immune cell subsets, including CD4+ or CD8+ T cell subsets (Figures 6A–6D) and NK cells (Figures 6E–6F), was assessed by flow cytometry in peripheral blood at baseline, day 14, and day 28 after IV administration of variant 20. At baseline, registered study mice had a mean of 62% hCD45, 5% CD3, and 7% CD56. Figure 6A shows the frequency (cells / μL) of KIR+ hCD8 T cells. Figure 6B shows the frequency (cells / μL) of hCD4 T cells. Figure 6C shows the frequency (cells / μL) of hCD8 T cells. Figure 6D shows the ratio of CD4 T cells to CD8 T cells. Figure 6E shows the frequency of hCD56+ NK cells (cells / μL). Figure 6F shows the frequency of KIR+ hCD56+ NK cells (cells / μL). [Figure 6D] Figures 6A–6F show the frequency of human immune cells in the peripheral blood of huCD34+NSG-Tg(Hu-IL15) mice after a single dose of variant 20. The frequency of immune cell subsets, including CD4+ or CD8+ T cell subsets (Figures 6A–6D) and NK cells (Figures 6E–6F), was assessed by flow cytometry in peripheral blood at baseline, day 14, and day 28 after IV administration of variant 20. At baseline, registered study mice had a mean of 62% hCD45, 5% CD3, and 7% CD56. Figure 6A shows the frequency (cells / μL) of KIR+ hCD8 T cells. Figure 6B shows the frequency (cells / μL) of hCD4 T cells. Figure 6C shows the frequency (cells / μL) of hCD8 T cells. Figure 6D shows the ratio of CD4 T cells to CD8 T cells. Figure 6E shows the frequency of hCD56+ NK cells (cells / μL). Figure 6F shows the frequency of KIR+ hCD56+ NK cells (cells / μL). [Figure 6E]Figures 6A–6F show the frequency of human immune cells in the peripheral blood of huCD34+NSG-Tg(Hu-IL15) mice after a single dose of variant 20. The frequency of immune cell subsets, including CD4+ or CD8+ T cell subsets (Figures 6A–6D) and NK cells (Figures 6E–6F), was assessed by flow cytometry in peripheral blood at baseline, day 14, and day 28 after IV administration of variant 20. At baseline, registered study mice had a mean of 62% hCD45, 5% CD3, and 7% CD56. Figure 6A shows the frequency (cells / μL) of KIR+ hCD8 T cells. Figure 6B shows the frequency (cells / μL) of hCD4 T cells. Figure 6C shows the frequency (cells / μL) of hCD8 T cells. Figure 6D shows the ratio of CD4 T cells to CD8 T cells. Figure 6E shows the frequency of hCD56+ NK cells (cells / μL). Figure 6F shows the frequency of KIR+ hCD56+ NK cells (cells / μL). [Figure 6F] Figures 6A–6F show the frequency of human immune cells in the peripheral blood of huCD34+NSG-Tg(Hu-IL15) mice after a single dose of variant 20. The frequency of immune cell subsets, including CD4+ or CD8+ T cell subsets (Figures 6A–6D) and NK cells (Figures 6E–6F), was assessed by flow cytometry in peripheral blood at baseline, day 14, and day 28 after IV administration of variant 20. At baseline, registered study mice had a mean of 62% hCD45, 5% CD3, and 7% CD56. Figure 6A shows the frequency (cells / μL) of KIR+ hCD8 T cells. Figure 6B shows the frequency (cells / μL) of hCD4 T cells. Figure 6C shows the frequency (cells / μL) of hCD8 T cells. Figure 6D shows the ratio of CD4 T cells to CD8 T cells. Figure 6E shows the frequency of hCD56+ NK cells (cells / μL). Figure 6F shows the frequency of KIR+ hCD56+ NK cells (cells / μL).

[0020] [Figure 7A]Figures 7A–7D show the binding of variant 20 to peripheral blood cells and spleen in huCD34+NSG-Tg(Hu-IL15) mice. The upper panel shows prevalence (Fc detected, as %Fc positive), and the lower panel shows MFI of %Fc positive cells. Data are presented for individuals with mean bar ± SD. Figure 7A shows the binding of variant 20 to all CD8 cells in peripheral blood. Figure 7B shows the binding of variant 20 to KIR+CD8 Treg cells in peripheral blood. Figure 7C shows the binding of variant 20 to NK cells in peripheral blood. Figure 7D shows a summary of the prevalence and MFI of variant 20 binding to all CD8 T cells, KIR+CD8, and NK cells in the spleen at the final time point. [Figure 7B] Figures 7A–7D show the binding of variant 20 to peripheral blood cells and spleen in huCD34+NSG-Tg(Hu-IL15) mice. The upper panel shows prevalence (Fc detected, as %Fc positive), and the lower panel shows MFI of %Fc positive cells. Data are presented for individuals with mean bar ± SD. Figure 7A shows the binding of variant 20 to all CD8 cells in peripheral blood. Figure 7B shows the binding of variant 20 to KIR+CD8 Treg cells in peripheral blood. Figure 7C shows the binding of variant 20 to NK cells in peripheral blood. Figure 7D shows a summary of the prevalence and MFI of variant 20 binding to all CD8 T cells, KIR+CD8, and NK cells in the spleen at the final time point. [Figure 7C] Figures 7A–7D show the binding of variant 20 to peripheral blood cells and spleen in huCD34+NSG-Tg(Hu-IL15) mice. The upper panel shows prevalence (Fc detected, as %Fc positive), and the lower panel shows MFI of %Fc positive cells. Data are presented for individuals with mean bar ± SD. Figure 7A shows the binding of variant 20 to all CD8 cells in peripheral blood. Figure 7B shows the binding of variant 20 to KIR+CD8 Treg cells in peripheral blood. Figure 7C shows the binding of variant 20 to NK cells in peripheral blood. Figure 7D shows a summary of the prevalence and MFI of variant 20 binding to all CD8 T cells, KIR+CD8, and NK cells in the spleen at the final time point. [Figure 7D] Figures 7A–7D show the binding of variant 20 to peripheral blood cells and spleen in huCD34+NSG-Tg(Hu-IL15) mice. The upper panel shows prevalence (Fc detected, as %Fc positive), and the lower panel shows MFI of %Fc positive cells. Data are presented for individuals with mean bar ± SD. Figure 7A shows the binding of variant 20 to all CD8 cells in peripheral blood. Figure 7B shows the binding of variant 20 to KIR+CD8 Treg cells in peripheral blood. Figure 7C shows the binding of variant 20 to NK cells in peripheral blood. Figure 7D shows a summary of the prevalence and MFI of variant 20 binding to all CD8 T cells, KIR+CD8, and NK cells in the spleen at the final time point.

[0021] [Figure 8A] Figures 8A to 8D show the pharmacological effects of a single dose of variant 20 in huCD34+NSG(IL-15Tg) mice. Figure 8A shows Ki67 MFI in all CD8 T cells. Figure 8B shows Ki67 MFI in KIR+CD8 T cells. Figure 8C shows the percentage of CD69+CD25+ cells in all CD8 T cells. Figure 8D shows the percentage of CD69+CD25+ cells in KIR+CD8 T cells. [Figure 8B] Figures 8A to 8D show the pharmacological effects of a single dose of variant 20 in huCD34+NSG(IL-15Tg) mice. Figure 8A shows Ki67 MFI in all CD8 T cells. Figure 8B shows Ki67 MFI in KIR+CD8 T cells. Figure 8C shows the percentage of CD69+CD25+ cells in all CD8 T cells. Figure 8D shows the percentage of CD69+CD25+ cells in KIR+CD8 T cells. [Figure 8C]Figures 8A to 8D show the pharmacological effects of a single dose of variant 20 in huCD34+NSG(IL-15Tg) mice. Figure 8A shows Ki67 MFI in all CD8 T cells. Figure 8B shows Ki67 MFI in KIR+CD8 T cells. Figure 8C shows the percentage of CD69+CD25+ cells in all CD8 T cells. Figure 8D shows the percentage of CD69+CD25+ cells in KIR+CD8 T cells. [Figure 8D] Figures 8A to 8D show the pharmacological effects of a single dose of variant 20 in huCD34+NSG(IL-15Tg) mice. Figure 8A shows Ki67 MFI in all CD8 T cells. Figure 8B shows Ki67 MFI in KIR+CD8 T cells. Figure 8C shows the percentage of CD69+CD25+ cells in all CD8 T cells. Figure 8D shows the percentage of CD69+CD25+ cells in KIR+CD8 T cells.

[0022] [Figure 9A] Figures 9A and 9B show the loss of CD69+ CD4 T cells after a single dose of variant 20. Figure 9A shows the percentage of total CD4 T cells. Figure 9B shows the percentage of activated (CD69+) CD4 T cells. [Figure 9B] Figures 9A and 9B show the loss of CD69+ CD4 T cells after a single dose of variant 20. Figure 9A shows the percentage of total CD4 T cells. Figure 9B shows the percentage of activated (CD69+) CD4 T cells.

[0023] [Figure 10]Figure 10 shows the time course of variant 20 (ng / mL) concentrations in blood samples derived from humanized CD34+NSG-Tg(Hu-IL15) mice. Variant 20 was detectable for 672 hours in 10 or 1 mg / kg CD34+NSG-Tg(Hu-IL15) mice (sequential microsamples) and 5 mg / kg BALB / cJ mice (serum), and was consistent between donors and strains. Serum samples from BALB / cJ mice were collected at n=3 per time point. Data are presented as the mean + / -SD of individuals at each time point shown in the graph. Square = 5 mg / kg BALB / cJ; inverted triangle = 10 mg / kg CD34+NSG-Tg(Hu-IL15); triangle = 1 mg / kg CD34+NSG-Tg(Hu-IL15).

[0024] [Figure 11] Figure 11 shows the frequency of CD8 Tregs as the percentage of total CD8 T cells in the peripheral blood of healthy donors and donors with rheumatic autoimmune disorders, where CD8 Tregs are defined as KIR2DL1 / 2 / 3 expressing (KIR+) CD8 Tregs. The frequency of CD8 Tregs is shown in healthy donors and in patients with psoriasis (left), systemic lupus erythematosus (SLE; center), psoriatic arthritis (PsA; center), ankylosing spondylitis (ASp; right), and Sjögren's syndrome (SS; right).

[0025] [Figure 12A]Figures 12A-12C show the upregulation of granzyme B in CD8 Tregs (CD8 Tregs expressing KIR2DL1 / 2 / 3) from donors with rheumatic autoimmune disorders after activation with anti-CD3 antibodies. PBMCs from healthy donors or patients with rheumatic autoimmune disorders were activated with anti-CD3 antibodies (OKT3), and intracellular granzyme B expression was subsequently evaluated by intracellular staining. PBMCs that were not activated after overnight rest were used as controls. The frequency of granzyme B-expressing CD8 Tregs as a percentage of total CD8 Tregs is shown for healthy donors and patients with psoriasis (Figure 12A), systemic lupus erythematosus (SLE; Figure 12B), psoriatic arthritis (PsA; Figure 12B), ankylosing spondylitis (ASp; Figure 12C), and Sjögren's syndrome (SS; Figure 12C) in both unstimulated and stimulated cells. [Figure 12B] Figures 12A-12C show the upregulation of granzyme B in CD8 Tregs (CD8 Tregs expressing KIR2DL1 / 2 / 3) from donors with rheumatic autoimmune disorders after activation with anti-CD3 antibodies. PBMCs from healthy donors or patients with rheumatic autoimmune disorders were activated with anti-CD3 antibodies (OKT3), and intracellular granzyme B expression was subsequently evaluated by intracellular staining. PBMCs that were not activated after overnight rest were used as controls. The frequency of granzyme B-expressing CD8 Tregs as a percentage of total CD8 Tregs is shown for healthy donors and patients with psoriasis (Figure 12A), systemic lupus erythematosus (SLE; Figure 12B), psoriatic arthritis (PsA; Figure 12B), ankylosing spondylitis (ASp; Figure 12C), and Sjögren's syndrome (SS; Figure 12C) in both unstimulated and stimulated cells. [Figure 12C]Figures 12A-12C show the upregulation of granzyme B in CD8 Tregs (CD8 Tregs expressing KIR2DL1 / 2 / 3) from donors with rheumatic autoimmune disorders after activation with anti-CD3 antibodies. PBMCs from healthy donors or patients with rheumatic autoimmune disorders were activated with anti-CD3 antibodies (OKT3), and intracellular granzyme B expression was subsequently evaluated by intracellular staining. PBMCs that were not activated after overnight rest were used as controls. The frequency of granzyme B-expressing CD8 Tregs as a percentage of total CD8 Tregs is shown for healthy donors and patients with psoriasis (Figure 12A), systemic lupus erythematosus (SLE; Figure 12B), psoriatic arthritis (PsA; Figure 12B), ankylosing spondylitis (ASp; Figure 12C), and Sjögren's syndrome (SS; Figure 12C) in both unstimulated and stimulated cells.

[0026] [Figure 13A] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13B] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13C] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13D-E] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13F-G] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13H-I]Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13J-L] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice. [Figure 13M-O] Figures 13A to 13O show the design and results of a multi-dose tolerability study in which variant 20 was administered to humanized CD34+NSG-Tg(Hu-IL15) mice.

[0027] In the repeated dose-toxicity study shown in Figure 13A, human CD34+ umbilical cord blood cells from two independent donors were transplanted into female NSG-Tg(Hu-IL15) mice and screened for inclusion in the study for 12 weeks. Figure 13B shows the percentages of hCD45, CD3 T, and CD56 NK cells at 12 weeks post-engraft in mice enrolled in the study. CD34+ cells from two donors were transplanted into 4-week-old NSG-Tg(Hu-IL15) mice, followed by evaluation of hCD45+, CD19+, CD3+, CD33+, and CD56+ cells at 12 weeks post-engraft. Mice with over 25% hCD45, over 3% hCD3, and over 2% hCD56 were accepted into the study. After shipment and acclimatization, animals received weekly IV doses of 5 or 50 mg / kg of Variant 20 or vehicle (approximately 17-18 weeks post-engraft). Blood samples were collected at specific post-dose time points from a subset of animals for PK (n=3 / dose / donor), serum cytokine analysis (n=3 / dose / donor), and immunophenotyping (n=5 per donor in each dose group). PK time points were collected pre-administration on days 1 and 22, and at 0.5, 2, 24, 96, and 168 hours post-administration. Post-administration time points were also collected on days 8, 15, and 29. Flow cytometry time points were pre-administration on days 1 and 15, and post-administration on day 29. Serum cytokine time points were pre-administration on day 1, and at 8 and 24 hours post-administration. Terminal blood and spleen samples were collected on day 29.

[0028] Body weight was measured during the study period (Figure 13C).

[0029] The frequencies of human immune cell subsets were assessed at baseline, day 15, and day 29 in peripheral blood and spleen after weekly IV administration of vehicle (Figures 13D-13O, left circle for each day), 50 mg / kg variant 20 (Figures 13D-13O, center circle for each day), or 5 mg / kg variant 20 (Figures 13D-13O, right circle for each day). Throughout the study, the percentages of total hCD45, CD3, CD4, CD8, NK (CD56+CD8-), KIR+CD8+, and KIR+NK cells in blood and spleen remained similar among 0 mg / kg (vehicle), 5 mg / kg variant 20, and 50 mg / kg variant 20 (Figures 13D-13I, for blood; Figures 13J-13O, for spleen; data are presented for individuals with mean bar ± SD).

[0030] [Figure 14A]Figures 14A-14I show the binding of variant 20 to cells in humanized CD34+NSG-Tg(Hu-IL15) mice from the toxicity study shown in Figure 13A. The binding of variant 20 to CD8 Treg (Figure 14A) and CD8 T cells (Figure 14B) from the peripheral blood of humanized CD34+NSG-Tg(Hu-IL15) mice before administration on day 15 (7 days after administration on day 8) and 30 minutes after administration on day 29 was evaluated by Fc detection using anti-human IgG1 Fc secondary antibody (vehicle = left circle for each day; variant 20 at 50 mg / kg = center circle for each day; variant 20 at 5 mg / kg = right circle for each day). The binding of variant 20 to CD8 Treg, CD8 T cells, KIR+NK cells, and NK cells (from left to right each day) derived from the spleen of humanized CD34+NSG-Tg(Hu-IL15) mice on day 29 (30 minutes after administration) (Figure 14C) was evaluated by Fc detection using an anti-human IgG1 Fc secondary antibody. Further examples of variant 20 binding are shown in Figures 14D-14G (peripheral blood; vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day) and Figures 14H-14I (spleen). Data are presented for individuals with mean bar ± SD. [Figure 14B]Figures 14A-14I show the binding of variant 20 to cells in humanized CD34+NSG-Tg(Hu-IL15) mice from the toxicity study shown in Figure 13A. The binding of variant 20 to CD8 Treg (Figure 14A) and CD8 T cells (Figure 14B) from the peripheral blood of humanized CD34+NSG-Tg(Hu-IL15) mice before administration on day 15 (7 days after administration on day 8) and 30 minutes after administration on day 29 was evaluated by Fc detection using anti-human IgG1 Fc secondary antibody (vehicle = left circle for each day; variant 20 at 50 mg / kg = center circle for each day; variant 20 at 5 mg / kg = right circle for each day). The binding of variant 20 to CD8 Treg, CD8 T cells, KIR+NK cells, and NK cells (from left to right each day) derived from the spleen of humanized CD34+NSG-Tg(Hu-IL15) mice on day 29 (30 minutes after administration) (Figure 14C) was evaluated by Fc detection using an anti-human IgG1 Fc secondary antibody. Further examples of variant 20 binding are shown in Figures 14D-14G (peripheral blood; vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day) and Figures 14H-14I (spleen). Data are presented for individuals with mean bar ± SD. [Figure 14C]Figures 14A-14I show the binding of variant 20 to cells in humanized CD34+NSG-Tg(Hu-IL15) mice from the toxicity study shown in Figure 13A. The binding of variant 20 to CD8 Treg (Figure 14A) and CD8 T cells (Figure 14B) from the peripheral blood of humanized CD34+NSG-Tg(Hu-IL15) mice before administration on day 15 (7 days after administration on day 8) and 30 minutes after administration on day 29 was evaluated by Fc detection using anti-human IgG1 Fc secondary antibody (vehicle = left circle for each day; variant 20 at 50 mg / kg = center circle for each day; variant 20 at 5 mg / kg = right circle for each day). The binding of variant 20 to CD8 Treg, CD8 T cells, KIR+NK cells, and NK cells (from left to right each day) derived from the spleen of humanized CD34+NSG-Tg(Hu-IL15) mice on day 29 (30 minutes after administration) (Figure 14C) was evaluated by Fc detection using an anti-human IgG1 Fc secondary antibody. Further examples of variant 20 binding are shown in Figures 14D-14G (peripheral blood; vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day) and Figures 14H-14I (spleen). Data are presented for individuals with mean bar ± SD. [Figure 14D-E]Figures 14A-14I show the binding of variant 20 to cells in humanized CD34+NSG-Tg(Hu-IL15) mice from the toxicity study shown in Figure 13A. The binding of variant 20 to CD8 Treg (Figure 14A) and CD8 T cells (Figure 14B) from the peripheral blood of humanized CD34+NSG-Tg(Hu-IL15) mice before administration on day 15 (7 days after administration on day 8) and 30 minutes after administration on day 29 was evaluated by Fc detection using anti-human IgG1 Fc secondary antibody (vehicle = left circle for each day; variant 20 at 50 mg / kg = center circle for each day; variant 20 at 5 mg / kg = right circle for each day). The binding of variant 20 to CD8 Treg, CD8 T cells, KIR+NK cells, and NK cells (from left to right each day) derived from the spleen of humanized CD34+NSG-Tg(Hu-IL15) mice on day 29 (30 minutes after administration) (Figure 14C) was evaluated by Fc detection using an anti-human IgG1 Fc secondary antibody. Further examples of variant 20 binding are shown in Figures 14D-14G (peripheral blood; vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day) and Figures 14H-14I (spleen). Data are presented for individuals with mean bar ± SD. [Figure 14F-G]Figures 14A-14I show the binding of variant 20 to cells in humanized CD34+NSG-Tg(Hu-IL15) mice from the toxicity study shown in Figure 13A. The binding of variant 20 to CD8 Treg (Figure 14A) and CD8 T cells (Figure 14B) from the peripheral blood of humanized CD34+NSG-Tg(Hu-IL15) mice before administration on day 15 (7 days after administration on day 8) and 30 minutes after administration on day 29 was evaluated by Fc detection using anti-human IgG1 Fc secondary antibody (vehicle = left circle for each day; variant 20 at 50 mg / kg = center circle for each day; variant 20 at 5 mg / kg = right circle for each day). The binding of variant 20 to CD8 Treg, CD8 T cells, KIR+NK cells, and NK cells (from left to right each day) derived from the spleen of humanized CD34+NSG-Tg(Hu-IL15) mice on day 29 (30 minutes after administration) (Figure 14C) was evaluated by Fc detection using an anti-human IgG1 Fc secondary antibody. Further examples of variant 20 binding are shown in Figures 14D-14G (peripheral blood; vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day) and Figures 14H-14I (spleen). Data are presented for individuals with mean bar ± SD. [Figure 14H-I]Figures 14A-14I show the binding of variant 20 to cells in humanized CD34+NSG-Tg(Hu-IL15) mice from the toxicity study shown in Figure 13A. The binding of variant 20 to CD8 Treg (Figure 14A) and CD8 T cells (Figure 14B) from the peripheral blood of humanized CD34+NSG-Tg(Hu-IL15) mice before administration on day 15 (7 days after administration on day 8) and 30 minutes after administration on day 29 was evaluated by Fc detection using anti-human IgG1 Fc secondary antibody (vehicle = left circle for each day; variant 20 at 50 mg / kg = center circle for each day; variant 20 at 5 mg / kg = right circle for each day). The binding of variant 20 to CD8 Treg, CD8 T cells, KIR+NK cells, and NK cells (from left to right each day) derived from the spleen of humanized CD34+NSG-Tg(Hu-IL15) mice on day 29 (30 minutes after administration) (Figure 14C) was evaluated by Fc detection using an anti-human IgG1 Fc secondary antibody. Further examples of variant 20 binding are shown in Figures 14D-14G (peripheral blood; vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day) and Figures 14H-14I (spleen). Data are presented for individuals with mean bar ± SD.

[0031] [Figure 15A-B]Figures 15A-15G show the effects of variant 20 on the activation and proliferation of cells derived from humanized CD34+NSG-Tg(Hu-IL15) mice, as shown in toxicity studies in Figures 13A-13B. Figure 15A shows the frequency of IFNγ+CD8 Treg cells, and Figure 15B shows the frequency of IFNγ+NK cells (vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day). Figure 15C shows the frequency of Helios+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15D shows the frequency of Ki67+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15E shows CD69+CD25+ activation in peripheral blood, KIR+CD8 T cells (upper panel) and KIR+NK cells (lower panel) (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). Figure 15F shows CD69+CD25+ activation in spleen cells (left to right: CD8 Treg, total CD8, KIR+NK cells, NK cells). Figure 15G shows CD69+ activation in all CD4 T cells (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). [Figure 15C-D]Figures 15A-15G show the effects of variant 20 on the activation and proliferation of cells derived from humanized CD34+NSG-Tg(Hu-IL15) mice, as shown in toxicity studies in Figures 13A-13B. Figure 15A shows the frequency of IFNγ+CD8 Treg cells, and Figure 15B shows the frequency of IFNγ+NK cells (vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day). Figure 15C shows the frequency of Helios+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15D shows the frequency of Ki67+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15E shows CD69+CD25+ activation in peripheral blood, KIR+CD8 T cells (upper panel) and KIR+NK cells (lower panel) (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). Figure 15F shows CD69+CD25+ activation in spleen cells (left to right: CD8 Treg, total CD8, KIR+NK cells, NK cells). Figure 15G shows CD69+ activation in all CD4 T cells (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). [Figure 15E]Figures 15A-15G show the effects of variant 20 on the activation and proliferation of cells derived from humanized CD34+NSG-Tg(Hu-IL15) mice, as shown in toxicity studies in Figures 13A-13B. Figure 15A shows the frequency of IFNγ+CD8 Treg cells, and Figure 15B shows the frequency of IFNγ+NK cells (vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day). Figure 15C shows the frequency of Helios+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15D shows the frequency of Ki67+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15E shows CD69+CD25+ activation in peripheral blood, KIR+CD8 T cells (upper panel) and KIR+NK cells (lower panel) (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). Figure 15F shows CD69+CD25+ activation in spleen cells (left to right: CD8 Treg, total CD8, KIR+NK cells, NK cells). Figure 15G shows CD69+ activation in all CD4 T cells (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). [Figure 15F]Figures 15A-15G show the effects of variant 20 on the activation and proliferation of cells derived from humanized CD34+NSG-Tg(Hu-IL15) mice, as shown in toxicity studies in Figures 13A-13B. Figure 15A shows the frequency of IFNγ+CD8 Treg cells, and Figure 15B shows the frequency of IFNγ+NK cells (vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day). Figure 15C shows the frequency of Helios+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15D shows the frequency of Ki67+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15E shows CD69+CD25+ activation in peripheral blood, KIR+CD8 T cells (upper panel) and KIR+NK cells (lower panel) (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). Figure 15F shows CD69+CD25+ activation in spleen cells (left to right: CD8 Treg, total CD8, KIR+NK cells, NK cells). Figure 15G shows CD69+ activation in all CD4 T cells (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). [Figure 15G]Figures 15A-15G show the effects of variant 20 on the activation and proliferation of cells derived from humanized CD34+NSG-Tg(Hu-IL15) mice, as shown in toxicity studies in Figures 13A-13B. Figure 15A shows the frequency of IFNγ+CD8 Treg cells, and Figure 15B shows the frequency of IFNγ+NK cells (vehicle = left circle each day; variant 20 at 50 mg / kg = center circle each day; variant 20 at 5 mg / kg = right circle each day). Figure 15C shows the frequency of Helios+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15D shows the frequency of Ki67+CD8 Treg cells (left circle) and NK cells (right circle). Figure 15E shows CD69+CD25+ activation in peripheral blood, KIR+CD8 T cells (upper panel) and KIR+NK cells (lower panel) (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day). Figure 15F shows CD69+CD25+ activation in spleen cells (left to right: CD8 Treg, total CD8, KIR+NK cells, NK cells). Figure 15G shows CD69+ activation in all CD4 T cells (vehicle = left circle each day; 50 mg / kg variant 20 = center circle each day; 5 mg / kg variant 20 = right circle each day).

[0032] [Figure 16] Figure 16 shows pro-inflammatory serum cytokines after a single dose of 5 mg / kg variant 20 or 50 mg / kg variant 20 at 8 and 24 hours. Data are shown as mean bar ± SD. Variant 20 did not increase the expression of pro-inflammatory serum cytokines.

[0033] [Figure 17A]Figures 17A–17D show the pharmacokinetic characteristics of variant 20 in cynomolgus monkeys. A single dose of variant 20 at 5, 0.5, or 0.05 mg / kg was injected into the animals. Blood was collected at multiple time points and processed into serum. Figure 17A shows the serum levels of variant 20. Figure 17B shows the pharmacokinetic parameters calculated from the concentration-time data. The relationship between dose and Cmax and dose and AUC are shown in Figures 17C and 17D, respectively. [Figure 17B] Figures 17A–17D show the pharmacokinetic characteristics of variant 20 in cynomolgus monkeys. A single dose of variant 20 at 5, 0.5, or 0.05 mg / kg was injected into the animals. Blood was collected at multiple time points and processed into serum. Figure 17A shows the serum levels of variant 20. Figure 17B shows the pharmacokinetic parameters calculated from the concentration-time data. The relationship between dose and Cmax and dose and AUC are shown in Figures 17C and 17D, respectively. [Figure 17C-D] Figures 17A–17D show the pharmacokinetic characteristics of variant 20 in cynomolgus monkeys. A single dose of variant 20 at 5, 0.5, or 0.05 mg / kg was injected into the animals. Blood was collected at multiple time points and processed into serum. Figure 17A shows the serum levels of variant 20. Figure 17B shows the pharmacokinetic parameters calculated from the concentration-time data. The relationship between dose and Cmax and dose and AUC are shown in Figures 17C and 17D, respectively.

[0034] [Figure 18A]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The escalating number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphed for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity. [Figure 18B-C]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The escalating number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphed for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity. [Figure 18D]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The escalating number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphed for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity. [Figure 18E]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The escalating number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphed for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity. [Figure 18F]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The escalating number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphed for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity. [Figure 18G-H]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The incrementally increasing number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphs for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity. [Figure 18I]Figures 18A–18I describe the characteristics of CD8 Tregs and methods of their modulation. KIR CD8 Tregs express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac disease donor PBMCs, dose-dependently and specifically eliminate activated gliadin-responsive cell lines. Figures 18A and 18B show the CD8 Treg phenotypic markers NKG2C, Helios, and KLRG1 (Figure 18A) and the cell lysis marker granzyme B (Figure 18B) in healthy donor PBMCs seeded overnight in and without 1 ug / mL of anti-CD3, and then stained with KIR antibody. The presence of each of these markers is shown for KIR+ and KIR-CD8 T cell populations from seven healthy donors. Figure 18C shows a magnified view of the CD8 Tregs. KIR+CD8 Tregs were isolated from celiac disease PBMCs after expansion with IL-7 peptide, IL-15, and gliadin peptide. The selected CD8 Tregs were then placed with gliadin-enhanced CD4 T cells and autologous APCs for 3 days, with or without additional gliadin peptide stimulation (unstimulated), and the CD8 Treg expansion endpoint is shown. The results are representative of two independent experiments across three different celiac disease donors. In Figure 18D, celiac disease donor PBMCs were enriched with CD8 Tregs after 7 days of expansion and selection with IL-7 and IL-15. The escalating number of CD8 Treg-enriched cells were then placed with GFP+ activated LS2.8 SKW CD4+ target cells. The percentage change in GFP+ targets from 8 hours onwards is shown graphed for target cells in the absence of CD8 Tregs and with increasing CD8 Treg counts. The results are representative of three independent experiments. Figure 18E shows the percentage change in GFP+ targets over time (n=2 independent experiments) for CD8 Treg-enriched cells placed for 48 hours with either activated or inactivated parental SKW or α-gliadin peptide-specific SKW target cells. Figures 18F–18H show that CD8 Treg-enriched CD8 cells eliminate activated gliadin-responsive CD4 targets.CD8 T cells were expanded in IL-7 and IL-15 for 7 days, sorted with the surface marker KLRG1+CD244+CD28- (CD8 Treg enriched effector) or KLRG1-CD244-CD28+ (non-CD8 Treg effector), and then placed in a 2:1 ratio with activated gliadin-responsive GFP+ SKW CD4 target cell lines enriched from celiac disease donor PBMCs. The percentage change in GFP+ targets from 8 hours is shown for both CD8 Treg effector and non-CD8 Treg effector cells over 48 hours, showing only target cells without CD8 effector addition (Figure 18G, 18H). Incucyte generation videos show the decrease in masked GFP+ gliadin-responsive SKW target cells over 48 hours for both CD8 Treg enriched effector cells (Figure 18G) and non-CD8 Treg effector cells (Figure 18H). Figure 18I shows a schematic diagram for the modulation of CD8 Treg activity.

[0035] [Figure 19A]Figures 19A–19E show bispecific antibodies that bind to target surface receptors on CD8 Treg cells, demonstrating that binding is specific and dose-dependent. Figure 19A shows the binding affinity to human KIR2DL1 / 2 / 3 and CD8α. The table shows the measured association and dissociation rate constants for each binding interaction, as well as the KD binding affinity calculated from the measured rate constants. Figure 19B shows the dose-dependent binding curves for binding to KIR2DL1 / 2 / 3 and CD8α target antigens, along with the association and dissociation curves detected by Bio-Layer Interferometry. Figure 19C shows the dose-response curves of the bispecific antibody against stably expressed KIR2DL1 and CD8αSKW cell lines. EC50 values ​​were calculated for each cell line using Prism Graphpad software. The binding curves are representative of 14 independent experiments for the KIR2DL1 strain and 10 independent experiments for the CD8 strain. Figure 19D shows Ab00 binding to KIR3DL1-transfected HEK 293 or untransfected cells. Dose-dependent binding of the KIR3DL1-targeted bispecific antibody is shown as a positive control against KIR3DL1-transfected cell lines. Figure 19E shows a Retrogenix live-cell microarray technology study evaluating surface protein library binding of Ab20 and single-arm control to HEK 293 T cells. A summary report of targeted binding is provided. [Figure 19B-1]Figures 19A–19E show bispecific antibodies that bind to target surface receptors on CD8 Treg cells, demonstrating that binding is specific and dose-dependent. Figure 19A shows the binding affinity to human KIR2DL1 / 2 / 3 and CD8α. The table shows the measured association and dissociation rate constants for each binding interaction, as well as the KD binding affinity calculated from the measured rate constants. Figure 19B shows the dose-dependent binding curves for binding to KIR2DL1 / 2 / 3 and CD8α target antigens, along with the association and dissociation curves detected by Bio-Layer Interferometry. Figure 19C shows the dose-response curves of the bispecific antibody against stably expressed KIR2DL1 and CD8αSKW cell lines. EC50 values ​​were calculated for each cell line using Prism Graphpad software. The binding curves are representative of 14 independent experiments for the KIR2DL1 strain and 10 independent experiments for the CD8 strain. Figure 19D shows Ab00 binding to KIR3DL1-transfected HEK 293 or untransfected cells. Dose-dependent binding of the KIR3DL1-targeted bispecific antibody is shown as a positive control against KIR3DL1-transfected cell lines. Figure 19E shows a Retrogenix live-cell microarray technology study evaluating surface protein library binding of Ab20 and single-arm control to HEK 293 T cells. A summary report of targeted binding is provided. [Figure 19B-2]Figures 19A–19E show bispecific antibodies that bind to target surface receptors on CD8 Treg cells, demonstrating that binding is specific and dose-dependent. Figure 19A shows the binding affinity to human KIR2DL1 / 2 / 3 and CD8α. The table shows the measured association and dissociation rate constants for each binding interaction, as well as the KD binding affinity calculated from the measured rate constants. Figure 19B shows the dose-dependent binding curves for binding to KIR2DL1 / 2 / 3 and CD8α target antigens, along with the association and dissociation curves detected by Bio-Layer Interferometry. Figure 19C shows the dose-response curves of the bispecific antibody against stably expressed KIR2DL1 and CD8αSKW cell lines. EC50 values ​​were calculated for each cell line using Prism Graphpad software. The binding curves are representative of 14 independent experiments for the KIR2DL1 strain and 10 independent experiments for the CD8 strain. Figure 19D shows Ab00 binding to KIR3DL1-transfected HEK 293 or untransfected cells. Dose-dependent binding of the KIR3DL1-targeted bispecific antibody is shown as a positive control against KIR3DL1-transfected cell lines. Figure 19E shows a Retrogenix live-cell microarray technology study evaluating surface protein library binding of Ab20 and single-arm control to HEK 293 T cells. A summary report of targeted binding is provided. [Figure 19C]Figures 19A–19E show bispecific antibodies that bind to target surface receptors on CD8 Treg cells, demonstrating that binding is specific and dose-dependent. Figure 19A shows the binding affinity to human KIR2DL1 / 2 / 3 and CD8α. The table shows the measured association and dissociation rate constants for each binding interaction, as well as the KD binding affinity calculated from the measured rate constants. Figure 19B shows the dose-dependent binding curves for binding to KIR2DL1 / 2 / 3 and CD8α target antigens, along with the association and dissociation curves detected by Bio-Layer Interferometry. Figure 19C shows the dose-response curves of the bispecific antibody against stably expressed KIR2DL1 and CD8αSKW cell lines. EC50 values ​​were calculated for each cell line using Prism Graphpad software. The binding curves are representative of 14 independent experiments for the KIR2DL1 strain and 10 independent experiments for the CD8 strain. Figure 19D shows Ab00 binding to KIR3DL1-transfected HEK 293 or untransfected cells. Dose-dependent binding of the KIR3DL1-targeted bispecific antibody is shown as a positive control against KIR3DL1-transfected cell lines. Figure 19E shows a Retrogenix live-cell microarray technology study evaluating surface protein library binding of Ab20 and single-arm control to HEK 293 T cells. A summary report of targeted binding is provided. [Figure 19D]Figures 19A–19E show bispecific antibodies that bind to target surface receptors on CD8 Treg cells, demonstrating that binding is specific and dose-dependent. Figure 19A shows the binding affinity to human KIR2DL1 / 2 / 3 and CD8α. The table shows the measured association and dissociation rate constants for each binding interaction, as well as the KD binding affinity calculated from the measured rate constants. Figure 19B shows the dose-dependent binding curves for binding to KIR2DL1 / 2 / 3 and CD8α target antigens, along with the association and dissociation curves detected by Bio-Layer Interferometry. Figure 19C shows the dose-response curves of the bispecific antibody against stably expressed KIR2DL1 and CD8αSKW cell lines. EC50 values ​​were calculated for each cell line using Prism Graphpad software. The binding curves are representative of 14 independent experiments for the KIR2DL1 strain and 10 independent experiments for the CD8 strain. Figure 19D shows Ab00 binding to KIR3DL1-transfected HEK 293 or untransfected cells. Dose-dependent binding of the KIR3DL1-targeted bispecific antibody is shown as a positive control against KIR3DL1-transfected cell lines. Figure 19E shows a Retrogenix live-cell microarray technology study evaluating surface protein library binding of Ab20 and single-arm control to HEK 293 T cells. A summary report of targeted binding is provided. [Figure 19E]Figures 19A–19E show bispecific antibodies that bind to target surface receptors on CD8 Treg cells, demonstrating that binding is specific and dose-dependent. Figure 19A shows the binding affinity to human KIR2DL1 / 2 / 3 and CD8α. The table shows the measured association and dissociation rate constants for each binding interaction, as well as the KD binding affinity calculated from the measured rate constants. Figure 19B shows the dose-dependent binding curves for binding to KIR2DL1 / 2 / 3 and CD8α target antigens, along with the association and dissociation curves detected by Bio-Layer Interferometry. Figure 19C shows the dose-response curves of the bispecific antibody against stably expressed KIR2DL1 and CD8αSKW cell lines. EC50 values ​​were calculated for each cell line using Prism Graphpad software. The binding curves are representative of 14 independent experiments for the KIR2DL1 strain and 10 independent experiments for the CD8 strain. Figure 19D shows Ab00 binding to KIR3DL1-transfected HEK 293 or untransfected cells. Dose-dependent binding of the KIR3DL1-targeted bispecific antibody is shown as a positive control against KIR3DL1-transfected cell lines. Figure 19E shows a Retrogenix live-cell microarray technology study evaluating surface protein library binding of Ab20 and single-arm control to HEK 293 T cells. A summary report of targeted binding is provided.

[0036] [Figure 20A]Figures 20A–20D show specific binding by bispecific antibodies to target surface receptors on CD8 Tregs. Figure 20A shows simultaneous co-binding of antigens to bispecific antibodies as determined by bio-layer interferometry. The bispecific antibody was captured with either KIR2DL or CD8α, and further antigen binding was then detected with CD8α or KIR2DL, respectively. Figure 20B shows dose-dependent bispecific antibodies binding to the immune cell population within the whole human PBMC as detected by an anti-human Fc secondary antibody. Data are representative of seven independent experiments. Figure 20C shows quantification of CD8 and KIR binding sites per CD8 Treg target cell, determined based on receptor MFI and quantitative beads. Results are shown for CD8 Treg-derived PBMCs from 12 healthy donors, 3 celiac disease donors, and 5 Crohn's disease donors. Figure 20D shows the detection of unbound KIR sites at saturated doses of a fluorescently labeled single-arm KIR-Fc antibody on CD8 Tregs in total PBMCs after incubation with escalating doses of a bispecific antibody. IC50 values ​​for KIR-Fc binding were calculated using Prism Graphpad software and a nonlinear fit log(inhibitor) versus response with variable gradients and four parameters. The graph represents binding data across three independent healthy donors. [Figure 20B]Figures 20A–20D show specific binding by bispecific antibodies to target surface receptors on CD8 Tregs. Figure 20A shows simultaneous co-binding of antigens to bispecific antibodies as determined by bio-layer interferometry. The bispecific antibody was captured with either KIR2DL or CD8α, and further antigen binding was then detected with CD8α or KIR2DL, respectively. Figure 20B shows dose-dependent bispecific antibodies binding to the immune cell population within the whole human PBMC as detected by an anti-human Fc secondary antibody. Data are representative of seven independent experiments. Figure 20C shows quantification of CD8 and KIR binding sites per CD8 Treg target cell, determined based on receptor MFI and quantitative beads. Results are shown for CD8 Treg-derived PBMCs from 12 healthy donors, 3 celiac disease donors, and 5 Crohn's disease donors. Figure 20D shows the detection of unbound KIR sites at saturated doses of a fluorescently labeled single-arm KIR-Fc antibody on CD8 Tregs in total PBMCs after incubation with escalating doses of a bispecific antibody. IC50 values ​​for KIR-Fc binding were calculated using Prism Graphpad software and a nonlinear fit log(inhibitor) versus response with variable gradients and four parameters. The graph represents binding data across three independent healthy donors. [Figure 20C]Figures 20A–20D show specific binding by bispecific antibodies to target surface receptors on CD8 Tregs. Figure 20A shows simultaneous co-binding of antigens to bispecific antibodies as determined by bio-layer interferometry. The bispecific antibody was captured with either KIR2DL or CD8α, and further antigen binding was then detected with CD8α or KIR2DL, respectively. Figure 20B shows dose-dependent bispecific antibodies binding to the immune cell population within the whole human PBMC as detected by an anti-human Fc secondary antibody. Data are representative of seven independent experiments. Figure 20C shows quantification of CD8 and KIR binding sites per CD8 Treg target cell, determined based on receptor MFI and quantitative beads. Results are shown for CD8 Treg-derived PBMCs from 12 healthy donors, 3 celiac disease donors, and 5 Crohn's disease donors. Figure 20D shows the detection of unbound KIR sites at saturated doses of a fluorescently labeled single-arm KIR-Fc antibody on CD8 Tregs in total PBMCs after incubation with escalating doses of a bispecific antibody. IC50 values ​​for KIR-Fc binding were calculated using Prism Graphpad software and a nonlinear fit log(inhibitor) versus response with variable gradients and four parameters. The graph represents binding data across three independent healthy donors. [Figure 20D]Figures 20A–20D show specific binding by bispecific antibodies to target surface receptors on CD8 Tregs. Figure 20A shows simultaneous co-binding of antigens to bispecific antibodies as determined by bio-layer interferometry. The bispecific antibody was captured with either KIR2DL or CD8α, and further antigen binding was then detected with CD8α or KIR2DL, respectively. Figure 20B shows dose-dependent bispecific antibodies binding to the immune cell population within the whole human PBMC as detected by an anti-human Fc secondary antibody. Data are representative of seven independent experiments. Figure 20C shows quantification of CD8 and KIR binding sites per CD8 Treg target cell, determined based on receptor MFI and quantitative beads. Results are shown for CD8 Treg-derived PBMCs from 12 healthy donors, 3 celiac disease donors, and 5 Crohn's disease donors. Figure 20D shows the detection of unbound KIR sites at saturated doses of a fluorescently labeled single-arm KIR-Fc antibody on CD8 Tregs in total PBMCs after incubation with escalating doses of a bispecific antibody. IC50 values ​​for KIR-Fc binding were calculated using Prism Graphpad software and a nonlinear fit log(inhibitor) versus response with variable gradients and four parameters. The graph represents binding data across three independent healthy donors.

[0037] [Figure 21A-B]Figures 21A–21D show bispecific antibody activation of CD8 Tregs in celiac disease and healthy PBMCs. Figures 21A and 21B show the percentage of CD8 Tregs detected in PBMCs from healthy donors (n=2) and celiac disease donors (n=3) across the same donor in all PBMCs (Figure 21A) and in associated antibody dose-dependent CD8 Treg binding (Figure 21B). Figure 21C shows antibody dose-dependent activation of CD8 Tregs in celiac disease donor PBMCs across three different celiac disease donors, determined by the percentage of CD69 and ICOS-positive CD8 Tregs after 48 hours of incubation. Figure 21D shows that CD8 Treg activation is not induced by the control anti-RSV antibody. Healthy donor PBMCs were thawed overnight, and the following day, they were seeded with 100 nM Ab20 or anti-RSV control antibody for 48 hours. The graph shows the percentage of CD69-positive CD8 Tregs detected after incubation with no antibody, anti-RSV, or Ab20 across two different healthy donors. [Figure 21C]Figures 21A–21D show bispecific antibody activation of CD8 Tregs in celiac disease and healthy PBMCs. Figures 21A and 21B show the percentage of CD8 Tregs detected in PBMCs from healthy donors (n=2) and celiac disease donors (n=3) across the same donor in all PBMCs (Figure 21A) and in associated antibody dose-dependent CD8 Treg binding (Figure 21B). Figure 21C shows antibody dose-dependent activation of CD8 Tregs in celiac disease donor PBMCs across three different celiac disease donors, determined by the percentage of CD69 and ICOS-positive CD8 Tregs after 48 hours of incubation. Figure 21D shows that CD8 Treg activation is not induced by the control anti-RSV antibody. Healthy donor PBMCs were thawed overnight, and the following day, they were seeded with 100 nM Ab20 or anti-RSV control antibody for 48 hours. The graph shows the percentage of CD69-positive CD8 Tregs detected after incubation with no antibody, anti-RSV, or Ab20 across two different healthy donors. [Figure 21D]Figures 21A–21D show bispecific antibody activation of CD8 Tregs in celiac disease and healthy PBMCs. Figures 21A and 21B show the percentage of CD8 Tregs detected in PBMCs from healthy donors (n=2) and celiac disease donors (n=3) across the same donor in all PBMCs (Figure 21A) and in associated antibody dose-dependent CD8 Treg binding (Figure 21B). Figure 21C shows antibody dose-dependent activation of CD8 Tregs in celiac disease donor PBMCs across three different celiac disease donors, determined by the percentage of CD69 and ICOS-positive CD8 Tregs after 48 hours of incubation. Figure 21D shows that CD8 Treg activation is not induced by the control anti-RSV antibody. Healthy donor PBMCs were thawed overnight, and the following day, they were seeded with 100 nM Ab20 or anti-RSV control antibody for 48 hours. The graph shows the percentage of CD69-positive CD8 Tregs detected after incubation with no antibody, anti-RSV, or Ab20 across two different healthy donors.

[0038] [Figure 22A]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors. [Figure 22B]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors. [Figure 22C]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors. [Figure 22D]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors. [Figure 22E]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors. [Figure 22F]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors. [Figure 22G]Figures 22A-22G show the induction of gliadin-specific target cell lysis in the celiac disease T cell co-culture assay. Figure 22A shows the CD8 Treg prevalence, granzyme B, and degranulation of celiac disease donor CD8 Tregs after incubation with CD4 target cells (1:5) and APCs for 3 days in the presence or absence of 0.02 μg / ml anti-CD3 and Ab00 (100 nM). Figure 22B shows the concentrations of IFNγ, TNFα, and GMCSF detected after the same 3-day co-culture of CD8 Tregs and CD4 targets as in B, with and without the addition of Ab00 and anti-CD3. Figures 22C and 22D show the cytokine concentrations in PBMCs derived from two celiac disease donors. PBMCs were incubated for 48 hours with escalating concentrations of Ab00 (1, 10, or 100 nM) in the presence of a low dose of anti-CD3 (0.1 ug / mL) (left), and the endpoint annexin + CD4 is shown. The concentrations of pro-inflammatory cytokines (IL-2, TNFα, IFNγ, IL-6, and IL-17a) detected in the supernatant after 48-hour incubation with low-dose anti-CD3 in or without Ab00 are shown in the respective graphs for two different celiac disease donors. Figure 22E shows the percentage of CD8 Treg and CD8 Treg granzyme B and CD107a expression shown after treatment with bispecific antibody. Figure 22F shows the total T cell count, percentage of necrotic and apoptotic (dead) cells, CD25 MFI, and CD4 readout, including gliadin-stimulated CD4 production of IFNγ, after 3-day incubation with bispecific antibody, compared to an untreated control. Figure 22G shows the percentage reduction in IFNγ, TNFα, and GMCSF concentrations detected in the supernatant of co-cultures stimulated with gliadin peptide in the presence of Ab00, compared to an untreated control. For Figures 22E-22G, the results are representative of two independent experiments involving three different celiac disease donors.

[0039] [Figure 23A]Figures 23A–23D show that Ab20 restores the function of CD8 Tregs and reduces the antigen-induced pro-inflammatory response in Crohn's disease donor PBMCs and organoids. Figure 23A shows the presence of CD8 Tregs in the peripheral blood of healthy and Crohn's disease donors. In Figure 23B, CD8 Tregs were stained for the intracellular cytolysis marker granzyme B and the transcription factor Helios in PBMCs from six healthy and six Crohn's disease donors. Baseline expression of CD161, CXCR3, and CD39+ on CD4 T cells in PBMCs from eight healthy and eleven Crohn's disease donors. In Figure 23C, Crohn's disease PBMCs were incubated for 7 days with a mixture of bacterial flagellin and OmpC peptide, as well as IL-7 and IL-15 cytokines, in and without 100 nM Ab20. The percentage increase in granzyme B concentration detected in the supernatant on day 5 of the study and the percentage decrease in INFγ and TNFα on day 7 are shown for Ab20-treated donors compared to untreated donors in both responders and non-responders in the assay. The percentage decrease in proliferation, as determined by diluted CFSE, is also shown on day 7 in both responders and non-responders compared to untreated controls. Figure 23D shows Helios expression on CD4 T cells in PBMCs from Crohn's disease donors stimulated overnight with anti-CD3 (1 ug / mL) in the presence and absence of Ab20 (100 nM). Paired two-sided t-tests for untreated and Ab20-treated samples did not show statistical significance between values ​​across the six Crohn's disease donors. Figure 23D also shows the percentage decrease in antigen-induced epithelial cell death in both celiac disease and Crohn's disease donor-derived organoids after incubation with 100 nM Ab20. The results represent organoids generated from intestinal tissue from three independent celiac disease donors and two Crohn's disease donors. [Figure 23B]Figures 23A–23D show that Ab20 restores the function of CD8 Tregs and reduces the antigen-induced pro-inflammatory response in Crohn's disease donor PBMCs and organoids. Figure 23A shows the presence of CD8 Tregs in the peripheral blood of healthy and Crohn's disease donors. In Figure 23B, CD8 Tregs were stained for the intracellular cytolysis marker granzyme B and the transcription factor Helios in PBMCs from six healthy and six Crohn's disease donors. Baseline expression of CD161, CXCR3, and CD39+ on CD4 T cells in PBMCs from eight healthy and eleven Crohn's disease donors. In Figure 23C, Crohn's disease PBMCs were incubated for 7 days with a mixture of bacterial flagellin and OmpC peptide, as well as IL-7 and IL-15 cytokines, in and without 100 nM Ab20. The percentage increase in granzyme B concentration detected in the supernatant on day 5 of the study and the percentage decrease in INFγ and TNFα on day 7 are shown for Ab20-treated donors compared to untreated donors in both responders and non-responders in the assay. The percentage decrease in proliferation, as determined by diluted CFSE, is also shown on day 7 in both responders and non-responders compared to untreated controls. Figure 23D shows Helios expression on CD4 T cells in PBMCs from Crohn's disease donors stimulated overnight with anti-CD3 (1 ug / mL) in the presence and absence of Ab20 (100 nM). Paired two-sided t-tests for untreated and Ab20-treated samples did not show statistical significance between values ​​across the six Crohn's disease donors. Figure 23D also shows the percentage decrease in antigen-induced epithelial cell death in both celiac disease and Crohn's disease donor-derived organoids after incubation with 100 nM Ab20. The results represent organoids generated from intestinal tissue from three independent celiac disease donors and two Crohn's disease donors. [Figure 23C]Figures 23A–23D show that Ab20 restores the function of CD8 Tregs and reduces the antigen-induced pro-inflammatory response in Crohn's disease donor PBMCs and organoids. Figure 23A shows the presence of CD8 Tregs in the peripheral blood of healthy and Crohn's disease donors. In Figure 23B, CD8 Tregs were stained for the intracellular cytolysis marker granzyme B and the transcription factor Helios in PBMCs from six healthy and six Crohn's disease donors. Baseline expression of CD161, CXCR3, and CD39+ on CD4 T cells in PBMCs from eight healthy and eleven Crohn's disease donors. In Figure 23C, Crohn's disease PBMCs were incubated for 7 days with a mixture of bacterial flagellin and OmpC peptide, as well as IL-7 and IL-15 cytokines, in and without 100 nM Ab20. The percentage increase in granzyme B concentration detected in the supernatant on day 5 of the study and the percentage decrease in INFγ and TNFα on day 7 are shown for Ab20-treated donors compared to untreated donors in both responders and non-responders in the assay. The percentage decrease in proliferation, as determined by diluted CFSE, is also shown on day 7 in both responders and non-responders compared to untreated controls. Figure 23D shows Helios expression on CD4 T cells in PBMCs from Crohn's disease donors stimulated overnight with anti-CD3 (1 ug / mL) in the presence and absence of Ab20 (100 nM). Paired two-sided t-tests for untreated and Ab20-treated samples did not show statistical significance between values ​​across the six Crohn's disease donors. Figure 23D also shows the percentage decrease in antigen-induced epithelial cell death in both celiac disease and Crohn's disease donor-derived organoids after incubation with 100 nM Ab20. The results represent organoids generated from intestinal tissue from three independent celiac disease donors and two Crohn's disease donors. [Figure 23D]Figures 23A–23D show that Ab20 restores the function of CD8 Tregs and reduces the antigen-induced pro-inflammatory response in Crohn's disease donor PBMCs and organoids. Figure 23A shows the presence of CD8 Tregs in the peripheral blood of healthy and Crohn's disease donors. In Figure 23B, CD8 Tregs were stained for the intracellular cytolysis marker granzyme B and the transcription factor Helios in PBMCs from six healthy and six Crohn's disease donors. Baseline expression of CD161, CXCR3, and CD39+ on CD4 T cells in PBMCs from eight healthy and eleven Crohn's disease donors. In Figure 23C, Crohn's disease PBMCs were incubated for 7 days with a mixture of bacterial flagellin and OmpC peptide, as well as IL-7 and IL-15 cytokines, in and without 100 nM Ab20. The percentage increase in granzyme B concentration detected in the supernatant on day 5 of the study and the percentage decrease in INFγ and TNFα on day 7 are shown for Ab20-treated donors compared to untreated donors in both responders and non-responders in the assay. The percentage decrease in proliferation, as determined by diluted CFSE, is also shown on day 7 in both responders and non-responders compared to untreated controls. Figure 23D shows Helios expression on CD4 T cells in PBMCs from Crohn's disease donors stimulated overnight with anti-CD3 (1 ug / mL) in the presence and absence of Ab20 (100 nM). Paired two-sided t-tests for untreated and Ab20-treated samples did not show statistical significance between values ​​across the six Crohn's disease donors. Figure 23D also shows the percentage decrease in antigen-induced epithelial cell death in both celiac disease and Crohn's disease donor-derived organoids after incubation with 100 nM Ab20. The results represent organoids generated from intestinal tissue from three independent celiac disease donors and two Crohn's disease donors.

[0040] [Figure 24A]Figures 24A–24F show the effect of Ab20 on organoids derived from Crohn's disease donors. Figures 24A–24C show that Crohn's disease organoids maintain tissue structure and that the tissue contains relevant immune cells. Figure 24A shows a representative image of organoids derived from Crohn's disease intestinal tissue. Figure 24B shows the percentages of epithelial (EpCAM+), T, B, and NK cells present in both the blood and primary intestinal tissue (colon) of seven different Crohn's disease donors at baseline. The T cell graph shows the percentages of CD4 and CD8 T cells detected in the blood and colon tissue at baseline. Figure 24C shows the quantification of KIR2DL and CD8 receptors per CD8 Treg in the blood and colon, determined based on receptor MFI and quantitative beads. The results are representative of three independent Crohn's disease donors. Figure 24D shows Ab20 binding detected by anti-human IgG Fc antibody in Ab20-treated wells compared to organoid wells not treated with antibody ("Ag") (left). The percentage increase in CD8 Treg activation (CD25) is shown to be determined by comparison with the untreated control at the study endpoint. n=3 independent experiments. The decrease in antigen-induced CD4 expansion after Ab20 treatment is also shown across n=4 independent experiments of organoid cultures from celiac disease and Crohn's disease. Figure 24E shows the decrease in antigen-induced epithelial cell death and CD4 T cell expansion across organoid cultures from celiac disease and Crohn's disease after Ab20 treatment. Figure 24F shows the percentage decrease in IL-17a and GMCSF detected in organoid supernatant after Ab20 treatment in two independent celiac disease donors. [Figure 24B]Figures 24A–24F show the effect of Ab20 on organoids derived from Crohn's disease donors. Figures 24A–24C show that Crohn's disease organoids maintain tissue structure and that the tissue contains relevant immune cells. Figure 24A shows a representative image of organoids derived from Crohn's disease intestinal tissue. Figure 24B shows the percentages of epithelial (EpCAM+), T, B, and NK cells present in both blood and primary intestinal tissue (colon) of seven different Crohn's disease donors at baseline. The T cell graph shows the percentages of CD4 and CD8 T cells detected in blood and colon tissue at baseline. Figure 24C shows the quantification of KIR2DL and CD8 receptors per CD8 Treg in blood and colon, determined based on receptor MFI and quantitative beads. The results are representative of three independent Crohn's disease donors. Figure 24D shows Ab20 binding detected by anti-human IgG Fc antibody in Ab20-treated wells compared to organoid wells not treated with antibody ("Ag") (left). The percentage increase in CD8 Treg activation (CD25) is shown to be determined by comparison with the untreated control at the study endpoint. n=3 independent experiments. The decrease in antigen-induced CD4 expansion after Ab20 treatment is also shown across n=4 independent experiments of organoid cultures from celiac disease and Crohn's disease. Figure 24E shows the decrease in antigen-induced epithelial cell death and CD4 T cell expansion across organoid cultures from celiac disease and Crohn's disease after Ab20 treatment. Figure 24F shows the percentage decrease in IL-17a and GMCSF detected in organoid supernatant after Ab20 treatment in two independent celiac disease donors. [Figure 24C]Figures 24A–24F show the effect of Ab20 on organoids derived from Crohn's disease donors. Figures 24A–24C show that Crohn's disease organoids maintain tissue structure and that the tissue contains relevant immune cells. Figure 24A shows a representative image of organoids derived from Crohn's disease intestinal tissue. Figure 24B shows the percentages of epithelial (EpCAM+), T, B, and NK cells present in both the blood and primary intestinal tissue (colon) of seven different Crohn's disease donors at baseline. The T cell graph shows the percentages of CD4 and CD8 T cells detected in the blood and colon tissue at baseline. Figure 24C shows the quantification of KIR2DL and CD8 receptors per CD8 Treg in the blood and colon, determined based on receptor MFI and quantitative beads. The results are representative of three independent Crohn's disease donors. Figure 24D shows Ab20 binding detected by anti-human IgG Fc antibody in Ab20-treated wells compared to organoid wells not treated with antibody ("Ag") (left). The percentage increase in CD8 Treg activation (CD25) is shown to be determined by comparison with the untreated control at the study endpoint. n=3 independent experiments. The decrease in antigen-induced CD4 expansion after Ab20 treatment is also shown across n=4 independent experiments of organoid cultures from celiac disease and Crohn's disease. Figure 24E shows the decrease in antigen-induced epithelial cell death and CD4 T cell expansion across organoid cultures from celiac disease and Crohn's disease after Ab20 treatment. Figure 24F shows the percentage decrease in IL-17a and GMCSF detected in organoid supernatant after Ab20 treatment in two independent celiac disease donors. [Figure 24D]Figures 24A–24F show the effect of Ab20 on organoids derived from Crohn's disease donors. Figures 24A–24C show that Crohn's disease organoids maintain tissue structure and that the tissue contains relevant immune cells. Figure 24A shows a representative image of organoids derived from Crohn's disease intestinal tissue. Figure 24B shows the percentages of epithelial (EpCAM+), T, B, and NK cells present in both the blood and primary intestinal tissue (colon) of seven different Crohn's disease donors at baseline. The T cell graph shows the percentages of CD4 and CD8 T cells detected in the blood and colon tissue at baseline. Figure 24C shows the quantification of KIR2DL and CD8 receptors per CD8 Treg in the blood and colon, determined based on receptor MFI and quantitative beads. The results are representative of three independent Crohn's disease donors. Figure 24D shows Ab20 binding detected by anti-human IgG Fc antibody in Ab20-treated wells compared to organoid wells not treated with antibody ("Ag") (left). The percentage increase in CD8 Treg activation (CD25) is shown to be determined by comparison with the untreated control at the study endpoint. n=3 independent experiments. The decrease in antigen-induced CD4 expansion after Ab20 treatment is also shown across n=4 independent experiments of organoid cultures from celiac disease and Crohn's disease. Figure 24E shows the decrease in antigen-induced epithelial cell death and CD4 T cell expansion across organoid cultures from celiac disease and Crohn's disease after Ab20 treatment. Figure 24F shows the percentage decrease in IL-17a and GMCSF detected in organoid supernatant after Ab20 treatment in two independent celiac disease donors. [Figure 24E-F]Figures 24A–24F show the effect of Ab20 on organoids derived from Crohn's disease donors. Figures 24A–24C show that Crohn's disease organoids maintain tissue structure and that the tissue contains relevant immune cells. Figure 24A shows a representative image of organoids derived from Crohn's disease intestinal tissue. Figure 24B shows the percentages of epithelial (EpCAM+), T, B, and NK cells present in both the blood and primary intestinal tissue (colon) of seven different Crohn's disease donors at baseline. The T cell graph shows the percentages of CD4 and CD8 T cells detected in the blood and colon tissue at baseline. Figure 24C shows the quantification of KIR2DL and CD8 receptors per CD8 Treg in the blood and colon, determined based on receptor MFI and quantitative beads. The results are representative of three independent Crohn's disease donors. Figure 24D shows Ab20 binding detected by anti-human IgG Fc antibody in Ab20-treated wells compared to organoid wells not treated with antibody ("Ag") (left). The percentage increase in CD8 Treg activation (CD25) is shown to be determined by comparison with the untreated control at the study endpoint. n=3 independent experiments. The decrease in antigen-induced CD4 expansion after Ab20 treatment is also shown across n=4 independent experiments of organoid cultures from celiac disease and Crohn's disease. Figure 24E shows the decrease in antigen-induced epithelial cell death and CD4 T cell expansion across organoid cultures from celiac disease and Crohn's disease after Ab20 treatment. Figure 24F shows the percentage decrease in IL-17a and GMCSF detected in organoid supernatant after Ab20 treatment in two independent celiac disease donors.

[0041] [Figure 25A]Figures 25A and 25B show that Ab00 does not activate CD8 and NK cells, and that the immune response to microorganisms and viruses is maintained. In Figure 25A, IFNg was measured on day 5 in the supernatant of PBMC cultures from three healthy donors and three celiac disease donors who were restimulated after the first 13 days of expansion with CEFT, influenza HA, SARS-CoV-2, and AVV5, 6, 8 peptides (0, 0.1, and 1 ug / mL) and tetanus toxoid (5 ug / mL) in the presence and absence of Ab00. In Figure 25B, IFNg levels after 5 days of PBMC culture in the presence of SEB (100 ng / mL) are shown as a positive polyclonal stimulation control. For both figures, the graphs show IFNg levels for all six donors, and each point represents the mean of two technical replicates. The untreated and Ab00-treated values ​​were compared using one-way ANOVA, and p<0.05 was considered a statistically significant result. [Figure 25B] Figures 25A and 25B show that Ab00 does not activate CD8 and NK cells, and that the immune response to microorganisms and viruses is maintained. In Figure 25A, IFNg was measured on day 5 in the supernatant of PBMC cultures from three healthy donors and three celiac disease donors who were restimulated after the first 13 days of expansion with CEFT, influenza HA, SARS-CoV-2, and AVV5, 6, 8 peptides (0, 0.1, and 1 ug / mL) and tetanus toxoid (5 ug / mL) in the presence and absence of Ab00. In Figure 25B, IFNg levels after 5 days of PBMC culture in the presence of SEB (100 ng / mL) are shown as a positive polyclonal stimulation control. For both figures, the graphs show IFNg levels for all six donors, and each point represents the mean of two technical replicates. The untreated and Ab00-treated values ​​were compared using one-way ANOVA, and p<0.05 was considered a statistically significant result.

[0042] [Figure 26A-B]Figures 26A–26B show the activation of CD8 T cells (Figure 26A) or NK cells (Figure 26B) (determined by the percentage of CD25-positive cells) in two healthy donor PBMCs after 2-day incubation in the presence and absence of gradually increasing concentrations of KIR monoarm antibody, CD8 monoarm antibody, and Ab20. Pause graphs show readings in the absence of additional anti-CD3 stimulation (0.1 ug / mL), and activation graphs show results in the presence of additional anti-CD3 stimulation over 48 hours of incubation. Figures 26C–26G show that Ab20 binds to target cells in the final tissue and does not result in the production of pro-inflammatory cytokines. Figure 26C shows the percentage of T cells (CD3+) and NK cells (CD56+) in CD34+ NSG-Tg (Hu-IL-15) mice after 12 weeks of initial engraftment. Each symbol represents an individual engrafted mouse, totaling 24 per donor. Figure 26D shows that Ab20 was detected in serum along with an antibody-like half-like substance, and also on CD8 Tregs, and that 5 mg / kg did not induce immune cell activation in IL-15 transgenic human transplanted mice. Ab20 binding detected at each blood collection time by anti-human Fc secondary antibody. Percentages of Fc-positive cells are shown for 5 mg / kg Ab20, single-arm KIR, single-arm CD8, or CD8 Tregs, total CD8, and NK cells after treatment with KIR bivalent antibody or saline control. Data are representative of two samples of empty-pooled blood from three mice that received CD34 cells from two different donors at baseline, 3, 24, and 72 hours, as well as a sample from all six mice at 168 hours. Figure 26E shows the percentage of total Fc-positive CD8 Treg, total CD8, or NK cells detected in terminal spleens treated with Ab20, vehicle, single-arm KIR or CD8, and bivalent KIR antibody or OKT3 (0.5 mg / kg) at the final time point (168 hours). Each symbol in the graph represents one mouse, and each donor (n=2) is represented across three mice in each treatment group.Figure 26F shows the activation of CD8 Treg, total CD8, and NK cells, as determined by the percentage of positive CD69 and CD25 cells, over the course of a study of 6 mice at each time point after injection of 5 mg / kg Ab20, single-arm KIR or CD8 antibody, or bivalent KIR antibody, along with 0.5 mg / kg anti-CD3 (OKT3) control antibody. Figure 26G shows the serum levels of pro-inflammatory cytokines graphed for each of the mice (n=6) in the study at the listed time points and treatment groups. Anti-CD3 (OKT3) is shown as a positive control for pro-inflammatory cytokine production. [Figure 26C]Figures 26A–26B show the activation of CD8 T cells (Figure 26A) or NK cells (Figure 26B) (determined by the percentage of CD25-positive cells) in two healthy donor PBMCs after 2-day incubation in the presence and absence of gradually increasing concentrations of KIR monoarm antibody, CD8 monoarm antibody, and Ab20. Pause graphs show readings in the absence of additional anti-CD3 stimulation (0.1 ug / mL), and activation graphs show results in the presence of additional anti-CD3 stimulation over 48 hours of incubation. Figures 26C–26G show that Ab20 binds to target cells in the final tissue and does not result in the production of pro-inflammatory cytokines. Figure 26C shows the percentage of T cells (CD3+) and NK cells (CD56+) in CD34+ NSG-Tg (Hu-IL-15) mice after 12 weeks of initial engraftment. Each symbol represents an individual engrafted mouse, totaling 24 per donor. Figure 26D shows that Ab20 was detected in serum along with an antibody-like half-like substance, and also on CD8 Tregs, and that 5 mg / kg did not induce immune cell activation in IL-15 transgenic human transplanted mice. Ab20 binding detected at each blood collection time by anti-human Fc secondary antibody. Percentages of Fc-positive cells are shown for 5 mg / kg Ab20, single-arm KIR, single-arm CD8, or CD8 Tregs, total CD8, and NK cells after treatment with KIR bivalent antibody or saline control. Data are representative of two samples of empty-pooled blood from three mice that received CD34 cells from two different donors at baseline, 3, 24, and 72 hours, as well as a sample from all six mice at 168 hours. Figure 26E shows the percentage of total Fc-positive CD8 Treg, total CD8, or NK cells detected in terminal spleens treated with Ab20, vehicle, single-arm KIR or CD8, and bivalent KIR antibody or OKT3 (0.5 mg / kg) at the final time point (168 hours). Each symbol in the graph represents one mouse, and each donor (n=2) is represented across three mice in each treatment group.Figure 26F shows the activation of CD8 Treg, total CD8, and NK cells, as determined by the percentage of positive CD69 and CD25 cells, over the course of a study of 6 mice at each time point after injection of 5 mg / kg Ab20, single-arm KIR or CD8 antibody, or bivalent KIR antibody, along with 0.5 mg / kg anti-CD3 (OKT3) control antibody. Figure 26G shows the serum levels of pro-inflammatory cytokines graphed for each of the mice (n=6) in the study at the listed time points and treatment groups. Anti-CD3 (OKT3) is shown as a positive control for pro-inflammatory cytokine production. [Figure 26D]Figures 26A–26B show the activation of CD8 T cells (Figure 26A) or NK cells (Figure 26B) (determined by the percentage of CD25-positive cells) in two healthy donor PBMCs after 2-day incubation in the presence and absence of gradually increasing concentrations of KIR monoarm antibody, CD8 monoarm antibody, and Ab20. Pause graphs show readings in the absence of additional anti-CD3 stimulation (0.1 ug / mL), and activation graphs show results in the presence of additional anti-CD3 stimulation over 48 hours of incubation. Figures 26C–26G show that Ab20 binds to target cells in the final tissue and does not result in the production of pro-inflammatory cytokines. Figure 26C shows the percentage of T cells (CD3+) and NK cells (CD56+) in CD34+ NSG-Tg (Hu-IL-15) mice after 12 weeks of initial engraftment. Each symbol represents an individual engrafted mouse, totaling 24 per donor. Figure 26D shows that Ab20 was detected in serum along with an antibody-like half-like substance, and also on CD8 Tregs, and that 5 mg / kg did not induce immune cell activation in IL-15 transgenic human transplanted mice. Ab20 binding detected at each blood collection time by anti-human Fc secondary antibody. Percentages of Fc-positive cells are shown for 5 mg / kg Ab20, single-arm KIR, single-arm CD8, or CD8 Tregs, total CD8, and NK cells after treatment with KIR bivalent antibody or saline control. Data are representative of two samples of empty-pooled blood from three mice that received CD34 cells from two different donors at baseline, 3, 24, and 72 hours, as well as a sample from all six mice at 168 hours. Figure 26E shows the percentage of total Fc-positive CD8 Treg, total CD8, or NK cells detected in terminal spleens treated with Ab20, vehicle, single-arm KIR or CD8, and bivalent KIR antibody or OKT3 (0.5 mg / kg) at the final time point (168 hours). Each symbol in the graph represents one mouse, and each donor (n=2) is represented across three mice in each treatment group.Figure 26F shows the activation of CD8 Treg, total CD8, and NK cells, as determined by the percentage of positive CD69 and CD25 cells, over the course of a study of 6 mice at each time point after injection of 5 mg / kg Ab20, single-arm KIR or CD8 antibody, or bivalent KIR antibody, along with 0.5 mg / kg anti-CD3 (OKT3) control antibody. Figure 26G shows the serum levels of pro-inflammatory cytokines graphed for each of the mice (n=6) in the study at the listed time points and treatment groups. Anti-CD3 (OKT3) is shown as a positive control for pro-inflammatory cytokine production. [Figure 26E]Figures 26A–26B show the activation of CD8 T cells (Figure 26A) or NK cells (Figure 26B) (determined by the percentage of CD25-positive cells) in two healthy donor PBMCs after 2-day incubation in the presence and absence of gradually increasing concentrations of KIR monoarm antibody, CD8 monoarm antibody, and Ab20. Pause graphs show readings in the absence of additional anti-CD3 stimulation (0.1 ug / mL), and activation graphs show results in the presence of additional anti-CD3 stimulation over 48 hours of incubation. Figures 26C–26G show that Ab20 binds to target cells in the final tissue and does not result in the production of pro-inflammatory cytokines. Figure 26C shows the percentage of T cells (CD3+) and NK cells (CD56+) in CD34+ NSG-Tg (Hu-IL-15) mice after 12 weeks of initial engraftment. Each symbol represents an individual engrafted mouse, totaling 24 per donor. Figure 26D shows that Ab20 was detected in serum along with an antibody-like half-like substance, and also on CD8 Tregs, and that 5 mg / kg did not induce immune cell activation in IL-15 transgenic human transplanted mice. Ab20 binding detected at each blood collection time by anti-human Fc secondary antibody. Percentages of Fc-positive cells are shown for 5 mg / kg Ab20, single-arm KIR, single-arm CD8, or CD8 Tregs, total CD8, and NK cells after treatment with KIR bivalent antibody or saline control. Data are representative of two samples of empty-pooled blood from three mice that received CD34 cells from two different donors at baseline, 3, 24, and 72 hours, as well as a sample from all six mice at 168 hours. Figure 26E shows the percentage of total Fc-positive CD8 Treg, total CD8, or NK cells detected in terminal spleens treated with Ab20, vehicle, single-arm KIR or CD8, and bivalent KIR antibody or OKT3 (0.5 mg / kg) at the final time point (168 hours). Each symbol in the graph represents one mouse, and each donor (n=2) is represented across three mice in each treatment group.Figure 26F shows the activation of CD8 Treg, total CD8, and NK cells, as determined by the percentage of positive CD69 and CD25 cells, over the course of a study of 6 mice at each time point after injection of 5 mg / kg Ab20, single-arm KIR or CD8 antibody, or bivalent KIR antibody, along with 0.5 mg / kg anti-CD3 (OKT3) control antibody. Figure 26G shows the serum levels of pro-inflammatory cytokines graphed for each of the mice (n=6) in the study at the listed time points and treatment groups. Anti-CD3 (OKT3) is shown as a positive control for pro-inflammatory cytokine production. [Figure 26F]Figures 26A–26B show the activation of CD8 T cells (Figure 26A) or NK cells (Figure 26B) (determined by the percentage of CD25-positive cells) in two healthy donor PBMCs after 2-day incubation in the presence and absence of gradually increasing concentrations of KIR monoarm antibody, CD8 monoarm antibody, and Ab20. Pause graphs show readings in the absence of additional anti-CD3 stimulation (0.1 ug / mL), and activation graphs show results in the presence of additional anti-CD3 stimulation over 48 hours of incubation. Figures 26C–26G show that Ab20 binds to target cells in the final tissue and does not result in the production of pro-inflammatory cytokines. Figure 26C shows the percentage of T cells (CD3+) and NK cells (CD56+) in CD34+ NSG-Tg (Hu-IL-15) mice after 12 weeks of initial engraftment. Each symbol represents an individual engrafted mouse, totaling 24 per donor. Figure 26D shows that Ab20 was detected in serum along with an antibody-like half-like substance, and also on CD8 Tregs, and that 5 mg / kg did not induce immune cell activation in IL-15 transgenic human transplanted mice. Ab20 binding detected at each blood collection time by anti-human Fc secondary antibody. Percentages of Fc-positive cells are shown for 5 mg / kg Ab20, single-arm KIR, single-arm CD8, or CD8 Tregs, total CD8, and NK cells after treatment with KIR bivalent antibody or saline control. Data are representative of two samples of empty-pooled blood from three mice that received CD34 cells from two different donors at baseline, 3, 24, and 72 hours, as well as a sample from all six mice at 168 hours. Figure 26E shows the percentage of total Fc-positive CD8 Treg, total CD8, or NK cells detected in terminal spleens treated with Ab20, vehicle, single-arm KIR or CD8, and bivalent KIR antibody or OKT3 (0.5 mg / kg) at the final time point (168 hours). Each symbol in the graph represents one mouse, and each donor (n=2) is represented across three mice in each treatment group.Figure 26F shows the activation of CD8 Treg, total CD8, and NK cells, as determined by the percentage of positive CD69 and CD25 cells, over the course of a study of 6 mice at each time point after injection of 5 mg / kg Ab20, single-arm KIR or CD8 antibody, or bivalent KIR antibody, along with 0.5 mg / kg anti-CD3 (OKT3) control antibody. Figure 26G shows the serum levels of pro-inflammatory cytokines graphed for each of the mice (n=6) in the study at the listed time points and treatment groups. Anti-CD3 (OKT3) is shown as a positive control for pro-inflammatory cytokine production. [Figure 26G]Figures 26A–26B show the activation of CD8 T cells (Figure 26A) or NK cells (Figure 26B) (determined by the percentage of CD25-positive cells) in two healthy donor PBMCs after 2-day incubation in the presence and absence of gradually increasing concentrations of KIR monoarm antibody, CD8 monoarm antibody, and Ab20. Pause graphs show readings in the absence of additional anti-CD3 stimulation (0.1 ug / mL), and activation graphs show results in the presence of additional anti-CD3 stimulation over 48 hours of incubation. Figures 26C–26G show that Ab20 binds to target cells in the final tissue and does not result in the production of pro-inflammatory cytokines. Figure 26C shows the percentage of T cells (CD3+) and NK cells (CD56+) in CD34+ NSG-Tg (Hu-IL-15) mice after 12 weeks of initial engraftment. Each symbol represents an individual engrafted mouse, totaling 24 per donor. Figure 26D shows that Ab20 was detected in serum along with an antibody-like half-like substance, and also on CD8 Tregs, and that 5 mg / kg did not induce immune cell activation in IL-15 transgenic human transplanted mice. Ab20 binding detected at each blood collection time by anti-human Fc secondary antibody. Percentages of Fc-positive cells are shown for 5 mg / kg Ab20, single-arm KIR, single-arm CD8, or CD8 Tregs, total CD8, and NK cells after treatment with KIR bivalent antibody or saline control. Data are representative of two samples of empty-pooled blood from three mice that received CD34 cells from two different donors at baseline, 3, 24, and 72 hours, as well as a sample from all six mice at 168 hours. Figure 26E shows the percentage of total Fc-positive CD8 Treg, total CD8, or NK cells detected in terminal spleens treated with Ab20, vehicle, single-arm KIR or CD8, and bivalent KIR antibody or OKT3 (0.5 mg / kg) at the final time point (168 hours). Each symbol in the graph represents one mouse, and each donor (n=2) is represented across three mice in each treatment group.Figure 26F shows the activation of CD8 Treg, total CD8, and NK cells, as determined by the percentage of positive CD69 and CD25 cells, over the course of a study of 6 mice at each time point after injection of 5 mg / kg Ab20, single-arm KIR or CD8 antibody, or bivalent KIR antibody, along with 0.5 mg / kg anti-CD3 (OKT3) control antibody. Figure 26G shows the serum levels of pro-inflammatory cytokines graphed for each of the mice (n=6) in the study at the listed time points and treatment groups. Anti-CD3 (OKT3) is shown as a positive control for pro-inflammatory cytokine production.

[0043] [Figure 27A-B]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27C]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27D]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27E]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27F-G]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27H]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27I]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27J]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively. [Figure 27K]Figures 27A-27K show the effects of bispecific antibody binding, including selective binding, efficacy, mechanism of action, and dose escalation, in an acute human PBMC transplant NSG mouse model. Figure 27A shows the experimental design for the survival study. NSG mice were irradiated (0.75 Gy) and intravenously injected with 1e7 human PBMCS from healthy donor 3578 (haplotype HLA-DQ2.5). Antibody (2 mg / kg) or saline was injected every 7 days until day 28 of the study. Low-dose IL-2 (25,000 IU) was injected every other day from day 0 to day 10 of the study (n=20 / cohort). Figure 27B shows the percentage of human CD45 detected in lymphocytes in the blood of mice on day 14 of the study across the saline, antibody (Ab00), and IL-2 treatment groups in the survival study. Figure 27C shows antibody binding to CD4, CD8, and CD8 Treg populations detected by MFI of anti-human Fc antibodies in the blood on day 20 of the study, spleens of early excised mice across treatment groups on day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice), and stained frozen splenocytes after thawing of mice removed for weight loss or clinical endpoints after the end of the study (Ab and saline; n=6 mice). The data for day 20 of the study are representative of the 8 mice remaining in the antibody treatment group and the 6 mice remaining in the low-dose IL-2 group. Figure 27D shows the percentage of peripheral blood CD25 and ICOS-positive CD8 and CD8 Treg detected in peripheral blood on days 9 and 15 of the study across the saline, Ab, and low-dose IL-2 treatment groups. The data are representative of 10 individual mice in each treatment group on day 9 of the study and early excised mice on day 15 of the study. Granzyme B MFI is shown for CD8 T cells and CD8 Tregs in the remaining mice across antibody and low-dose IL-2 treatment groups on day 20 of the study. Figure 27E shows the percentage of granzyme and MFI in CD8 Tregs in peripheral blood (right) or splenocytes (left) of mice terminated early for the clinical endpoint in the antibody dose escalation study.Figure 27F shows the absolute number of CD25+CD4 T cells / µL in blood shown for early excised mice at day 15 of the study (Ab and saline; n=3 mice, IL-2; n=2 mice) across saline, Ab, and IL-2 treatment groups. MFI of Ki67 is also shown for early excised mice across treatment groups at day 15 of the study in peripheral blood. Figure 27G shows the survival curves of mice across each of the three treatment groups (saline, Ab, and low-dose IL-2). Data are representative of 18 mice per treatment group. Figure 27H shows serum concentrations of IFNγ at day 14 of the survival study (left) or at day 42 of the study with escalating doses of antibody (right) in mice with a clinical disease score of 3 or greater. The symbols in each graph represent measurements from one mouse. Figure 27I shows H&E staining of diseased intestinal tissue in anti-CD3 stimulated control (left) or Ab (right) treated mice. Clinical disease scores for Ab (green) compared to saline (black) or abatacept technical control (red) groups (n=6 / cohort). Figures 27J–27K show the results of a study using human NSG mice that were transplanted with 1e7 human PBMCs after donor irradiation (0.5 Gy) and intravenously administered antibodies at increasingly increasing doses every 7 days from day 0 to 21. Figure 27J shows antibody binding detected by MFI of anti-human Fc secondary antibody across increasingly increasing doses of antibody in each treatment group for CD4, CD8, and CD8 Treg (KLRG1+CD8 T cells) in the blood on day 14 of the study. The percentages of CD25-positive KLRG1+CD8 T cells and Helios-positive CD8 T cells are shown with increasing doses of antibody in the blood on days 14 and 42 of the study. Day 14 shows data from 8 mice per group, while day 42 shows the mice remaining at this final point. Figure 27K shows the granzyme B (MFI and percentage) of the total CD8 and KLRG1+CD8 T cell populations in peripheral blood and spleen of mice (total of n=3 mice per group) that completed the study on day 11, with increasing antibody dose. The percentages of CD25 and annexin-positive CD4 T cells are shown for the remaining mice on day 42 of the study in both endpoint blood and spleen cells with increasing antibody dose.Each symbol in the graph represents an individual mouse in the saline group and the antibody group, respectively.

[0044] [Figure 28A-B]Figures 28A–28H show the results of further studies confirming that the bispecific antibody Ab00 at a dose of 2 mg / kg enhances survival via a hypothetical mechanism of action, supporting long-term target engagement. Figures 28A–28D show the results of a small cohort survival study (n=4 mice / group) using human NSG mice transplanted with 1e7 human PBMCs derived from donor LZ0007 after irradiation (0.5 Gy) and administered intravenously with 2 mg / kg Ab00 or saline every 7 days from day 0 to day 21. Figure 28A shows engraftment as measured by the percentage of human CD45 detected in the blood of mice surviving in both the saline-treated and Ab00-treated groups. Figure 28B shows the binding of Ab00 detected by MFI with anti-human secondary Fc on CD8 Treg in the blood at 2 hours post-administration and day 14 of the study, and the MFI of granzymes. The percentage of CD25-positive CD8 Tregs is also shown for the saline and Ab00-treated groups in the blood at 2 hours post-administration on day 14 of the study (after Ab00 injections on days 0 and 7 of the study). Figure 28C shows that the percentage of Annexin+ and CD25+CD4 T cells is shown for the remaining mice in both the saline-treated and Ab00-treated groups. Figure 28D shows the survival curves of mice throughout the study and up to the end of the study on day 42 for each treatment group (n=4). Figure 28E shows the in vivo study design: human NSG mice transplanted with 1e7 human PBMCs derived from donor AC3004 after irradiation (0.75 Gy), and administered 2 mg / kg Ab00 or saline every 7 days starting on day 0 until day 70 of the study. Each cohort consisted of 8 mice. Figure 28F shows the engraftment level, determined by the percentage of human CD45 immune cells in peripheral blood on day 14 of the study, for both saline-treated and Ab00-treated mice. Figure 28G shows Ab00 binding, determined by anti-human Fc secondary antibody MFI, on CD4, CD8, and CD8 Treg subsets, pre-administration, on day 14 of the study. Figure 28H shows Ab00 binding to CD4, CD8, and CD8 Treg immune cell subsets in the blood of the remaining Ab00-treated mice on days 49 and 70 of the study.For all graphs, each symbol represents an individual mouse that is alive at that time. [Figure 28C-D]Figures 28A–28H show the results of further studies confirming that the bispecific antibody Ab00 at a dose of 2 mg / kg enhances survival via a hypothetical mechanism of action, supporting long-term target engagement. Figures 28A–28D show the results of a small cohort survival study (n=4 mice / group) using human NSG mice transplanted with 1e7 human PBMCs derived from donor LZ0007 after irradiation (0.5 Gy) and administered intravenously with 2 mg / kg Ab00 or saline every 7 days from day 0 to day 21. Figure 28A shows engraftment as measured by the percentage of human CD45 detected in the blood of mice surviving in both the saline-treated and Ab00-treated groups. Figure 28B shows the binding of Ab00 detected by MFI with anti-human secondary Fc on CD8 Treg in the blood at 2 hours post-administration and day 14 of the study, and the MFI of granzymes. The percentage of CD25-positive CD8 Tregs is also shown for the saline and Ab00-treated groups in the blood at 2 hours post-administration on day 14 of the study (after Ab00 injections on days 0 and 7 of the study). Figure 28C shows that the percentage of Annexin+ and CD25+CD4 T cells is shown for the remaining mice in both the saline-treated and Ab00-treated groups. Figure 28D shows the survival curves of mice throughout the study and up to the end of the study on day 42 for each treatment group (n=4). Figure 28E shows the in vivo study design: human NSG mice transplanted with 1e7 human PBMCs derived from donor AC3004 after irradiation (0.75 Gy), and administered 2 mg / kg Ab00 or saline every 7 days starting on day 0 until day 70 of the study. Each cohort consisted of 8 mice. Figure 28F shows the engraftment level, determined by the percentage of human CD45 immune cells in peripheral blood on day 14 of the study, for both saline-treated and Ab00-treated mice. Figure 28G shows Ab00 binding, determined by anti-human Fc secondary antibody MFI, on CD4, CD8, and CD8 Treg subsets, pre-administration, on day 14 of the study. Figure 28H shows Ab00 binding to CD4, CD8, and CD8 Treg immune cell subsets in the blood of the remaining Ab00-treated mice on days 49 and 70 of the study.For all graphs, each symbol represents an individual mouse that is alive at that time. [Figure 28E-F]Figures 28A–28H show the results of further studies confirming that the bispecific antibody Ab00 at a dose of 2 mg / kg enhances survival via a hypothetical mechanism of action, supporting long-term target engagement. Figures 28A–28D show the results of a small cohort survival study (n=4 mice / group) using human NSG mice transplanted with 1e7 human PBMCs derived from donor LZ0007 after irradiation (0.5 Gy) and administered intravenously with 2 mg / kg Ab00 or saline every 7 days from day 0 to day 21. Figure 28A shows engraftment as measured by the percentage of human CD45 detected in the blood of mice surviving in both the saline-treated and Ab00-treated groups. Figure 28B shows the binding of Ab00 detected by MFI with anti-human secondary Fc on CD8 Treg in the blood at 2 hours post-administration and day 14 of the study, and the MFI of granzymes. The percentage of CD25-positive CD8 Tregs is also shown for the saline and Ab00-treated groups in the blood at 2 hours post-administration on day 14 of the study (after Ab00 injections on days 0 and 7 of the study). Figure 28C shows that the percentage of Annexin+ and CD25+CD4 T cells is shown for the remaining mice in both the saline-treated and Ab00-treated groups. Figure 28D shows the survival curves of mice throughout the study and up to the end of the study on day 42 for each treatment group (n=4). Figure 28E shows the in vivo study design: human NSG mice transplanted with 1e7 human PBMCs derived from donor AC3004 after irradiation (0.75 Gy), and administered 2 mg / kg Ab00 or saline every 7 days starting on day 0 until day 70 of the study. Each cohort consisted of 8 mice. Figure 28F shows the engraftment level, determined by the percentage of human CD45 immune cells in peripheral blood on day 14 of the study, for both saline-treated and Ab00-treated mice. Figure 28G shows Ab00 binding, determined by anti-human Fc secondary antibody MFI, on CD4, CD8, and CD8 Treg subsets, pre-administration, on day 14 of the study. Figure 28H shows Ab00 binding to CD4, CD8, and CD8 Treg immune cell subsets in the blood of the remaining Ab00-treated mice on days 49 and 70 of the study.For all graphs, each symbol represents an individual mouse that is alive at that time. [Figure 28G-H]Figures 28A–28H show the results of further studies confirming that the bispecific antibody Ab00 at a dose of 2 mg / kg enhances survival via a hypothetical mechanism of action, supporting long-term target engagement. Figures 28A–28D show the results of a small cohort survival study (n=4 mice / group) using human NSG mice transplanted with 1e7 human PBMCs derived from donor LZ0007 after irradiation (0.5 Gy) and administered intravenously with 2 mg / kg Ab00 or saline every 7 days from day 0 to day 21. Figure 28A shows engraftment as measured by the percentage of human CD45 detected in the blood of mice surviving in both the saline-treated and Ab00-treated groups. Figure 28B shows the binding of Ab00 detected by MFI with anti-human secondary Fc on CD8 Treg in the blood at 2 hours post-administration and day 14 of the study, and the MFI of granzymes. The percentage of CD25-positive CD8 Tregs is also shown for the saline and Ab00-treated groups in the blood at 2 hours post-administration on day 14 of the study (after Ab00 injections on days 0 and 7 of the study). Figure 28C shows that the percentage of Annexin+ and CD25+CD4 T cells is shown for the remaining mice in both the saline-treated and Ab00-treated groups. Figure 28D shows the survival curves of mice throughout the study and up to the end of the study on day 42 for each treatment group (n=4). Figure 28E shows the in vivo study design: human NSG mice transplanted with 1e7 human PBMCs derived from donor AC3004 after irradiation (0.75 Gy), and administered 2 mg / kg Ab00 or saline every 7 days starting on day 0 until day 70 of the study. Each cohort consisted of 8 mice. Figure 28F shows the engraftment level, determined by the percentage of human CD45 immune cells in peripheral blood on day 14 of the study, for both saline-treated and Ab00-treated mice. Figure 28G shows Ab00 binding, determined by anti-human Fc secondary antibody MFI, on CD4, CD8, and CD8 Treg subsets, pre-administration, on day 14 of the study. Figure 28H shows Ab00 binding to CD4, CD8, and CD8 Treg immune cell subsets in the blood of the remaining Ab00-treated mice on days 49 and 70 of the study.For all graphs, each symbol represents an individual mouse that is alive at that time. [Modes for carrying out the invention]

[0045] Detailed explanation I. Glossary The following sections provide a detailed description of CD8-targeting antibodies or their antigen-binding fragments comprising binding domains and binding proteins, as well as related pharmaceutical compositions, methods for activating CD8+ T cells (including CD8+ regulatory T cells, "CD8+ Treg cells"), and methods for treating or preventing diseases (e.g., autoimmune diseases). Before describing this disclosure in more detail, definitions of certain terms used herein are provided. Additional definitions are provided throughout this disclosure.

[0046] Unless the context requires otherwise interpretation, the word “comprise,” and its variations such as “comprises” and “comprising,” should be interpreted throughout this specification and the claims in an open, comprehensive sense, i.e., “comprises, but not limited to.” “Consisting of” means excluding more trace elements of other components and substantial method steps disclosed herein, and in the case of amino acids or nucleic acid sequences, excluding additional amino acids or nucleotides, respectively. The term “consisting essentially of” limits the claims to specific materials or steps or to elements that do not substantially affect the fundamental characteristics of the claimed invention. For example, a composition essentially consisting of elements as defined herein does not exclude trace contaminants from isolation and purification methods, nor does it exclude pharmaceutically acceptable carriers such as phosphate-buffered saline or preservatives. Similarly, if a protein contains additional amino acids that contribute up to 20% of its length and do not substantially affect its activity, the protein is essentially derived from a particular amino acid sequence (e.g., changing the protein's activity by 50% or less). Embodiments defined by each of these transitional clauses are within the scope of the present invention.

[0047] In this specification, the term "about" means +20% of the indicated range, value, or structure unless otherwise specified.

[0048] Where used herein, the terms "a" and "an" should be understood to include "one" or "one or more" of the listed components unless otherwise specified.

[0049] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof, and may be used synonymously with "and / or".

[0050] As used herein, the terms “contains” and “possess” are to be used synonymously, and these terms and their variations are to be interpreted as non-restrictive.

[0051] The term "substantially" does not exclude "completely." For example, a composition that "substantially does not contain" Y does not have to contain Y completely. If necessary, the term "substantially" may be omitted from the definition provided herein.

[0052] "As needed" or "as required" means that the following elements, components, events, or circumstances may or may not occur, and that the description includes both cases in which they occur and cases in which they do not.

[0053] As used herein, “amino acids” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids.

[0054] As used herein, the terms “peptide,” “polypeptide,” and “protein,” and variations thereof, refer to molecules comprising at least two amino acids linked to one another by (normal or modified) peptide bonds. For example, a peptide, polypeptide, or protein may contain, or be composed of, multiple amino acids selected from 20 amino acids or amino acid analogs or mimics defined by the genetic code, each linked to one another by at least one peptide bond. A peptide, polypeptide, or protein may contain, or be composed of, L-amino acids and / or D-amino acids (or their analogs or mimics). The terms “peptide,” “polypeptide,” and “protein” also include “peptide mimics,” defined as peptide analogs containing non-peptidic structural elements, which can mimic or antagonize the biological effects of a native parent peptide. In certain embodiments, peptide mimics lack features such as enzymatically cleavable peptide bonds.

[0055] Peptides, polypeptides, or proteins may contain, or be composed of, amino acids other than the 20 amino acids defined by the genetic code, in addition to these amino acids. In certain embodiments, peptides, polypeptides, or proteins in the context of this disclosure may include amino acids modified by natural processes, such as post-translational maturation processes, or by chemical processes (e.g., synthetic processes), which are known in the art and include those described herein. Such modifications may appear anywhere in the polypeptide, for example, in the peptide backbone; in the amino acid chain; or at the carboxyl or amino terminus. Peptides or polypeptides may be branched after ubiquitination, etc., or may be cyclic with or without branching. The terms “peptide,” “polypeptide,” and “protein” also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide, or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of nucleotides or nucleotide derivatives, covalent fixation of lipids or lipid derivatives, covalent fixation of phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfation, or amino acid addition such as arginylation or ubiquitination.Such modifications are described in the literature (see Proteins Structure and Molecular Properties (1993) 2nd Ed., TECreighton, New York; Post-translational Covalent Modifications of Proteins (1983) BC Johnson, Ed., Academic Press, New York; Seifter et al., Meth. Enzymol. 182:626-646, 1990; Rattan et al., Ann NY Acad Sci 663:48-62, 1992). Therefore, the terms “peptide,” “polypeptide,” and “protein” may include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, etc. Variants of proteins, peptides, and polypeptides in this disclosure are also intended. In certain embodiments, variant proteins, peptides, and polypeptides include or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a defined or reference amino acid sequence described herein.

[0056] While the terms "protein" and "polypeptide" are often used in reference to relatively large polypeptides, the term "peptide" is often used in reference to small polypeptides; however, the use of these terms in the art is redundant. The terms "protein" and "polypeptide" are used interchangeably herein when referring to encoded gene products and their fragments. Furthermore, as used herein, "(poly)peptide" and "protein" may be used interchangeably with reference to polymers of amino acid residues, such as multiple amino acid monomers linked by peptide bonds.

[0057] A "nucleic acid molecule," "polynucleotide," or "nucleic acid" refers to a polymer compound containing covalently linked nucleotides, which may consist of natural subunits (e.g., purine bases or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, while pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilidates, and phosphoramidates.

[0058] Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA) including cDNA, genomic DNA, and synthetic DNA, all of which can be single-stranded or double-stranded. In the case of single-stranded nucleic acid molecules, the nucleic acid molecule can be a coding strand or a non-coding (antisense) strand. Polynucleotides (including oligonucleotides) and their fragments can be generated, for example, by polymerase chain reaction (PCR) or in vitro translation, or by ligation, cleavage, endonuclease activity, or exonuclease activity.

[0059] Nucleic acid molecules that encode an amino acid sequence contain all nucleotide sequences that encode the same amino acid sequence. Some versions of a nucleotide sequence may also contain introns to the extent that introns can be removed via cotranscriptional or posttranscriptional mechanisms. Different nucleotide sequences may encode the same amino acid sequence as a result of redundancy or degeneracy of the genetic code, by splicing, or both.

[0060] Variants of the nucleic acid molecules of this disclosure are also intended. The variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the nucleic acid molecules of the nucleic acid molecules of the definition or reference polynucleotide as defined herein, or hybridize to polynucleotides under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65–68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. The nucleic acid molecule variants retain the ability to encode binding domains having the functions described herein, such as specifically binding to a target molecule.

[0061] As used herein, the term “sequence variant” means any sequence having one or more changes compared to a reference sequence, the reference sequence being any publicly available sequence and / or any of the sequences disclosed herein, i.e., SEQ ID NOs: 1 to SEQ ID NOs: 137. Thus, the term “sequence variant” includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments, a sequence variant in the context of a nucleotide sequence is also a nucleotide sequence, while in certain embodiments of a sequence variant in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. As used herein, a “sequence variant” may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference sequence.

[0062] "Percent sequence identity" refers to the relationship between two or more sequences determined by comparing them. Methods for determining sequence identity can be designed to provide the best possible match between the sequences being compared. For example, sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences can be ignored for comparison purposes. Percent sequence identity as used herein is calculated over the length of the reference sequence unless otherwise specified. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using the BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in the BLAST program can be found in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). In the context of this disclosure, when sequence analysis software is used for analysis, it will be understood that the results of the analysis are based on the “default values” of the referenced program. “Default values” means any set of values ​​or parameters that are initially loaded into the software when it is first initialized.

[0063] In relation to nucleic acid (nucleotide) sequences, a “sequence variant” has a modified sequence in which one or more nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by their standard single-letter notation (A, C, G, or T). Due to the degeneracy of the genetic code, a “sequence variant” of a nucleotide sequence may or may not result in a change in the respective reference amino acid sequence, i.e., an “amino acid sequence variant.” In certain embodiments, a nucleotide sequence variant does not result in an amino acid sequence variant (e.g., a silent mutation). In some embodiments, a nucleotide sequence variant resulting in one or more “non-silent” mutations is intended. In some embodiments, the nucleotide sequence variants of the Disclosure encode amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence. The nucleotides and amino sequences disclosed herein also refer to codon-optimized versions of reference or wild-type nucleotides or amino acid sequences. In any of the embodiments described herein, the polynucleotides of the Disclosure may be codon-optimized for host cells containing the polynucleotide (see, for example, Scholten et al., Clin. Immunol. 119:135-145, 2006). Codon optimization can be performed using known techniques and tools, for example, using the GenScript® OptimumGene® tool or the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include partially codon-optimized sequences (i.e., at least one codon is optimized for expression in the host cell) and fully codon-optimized sequences.

[0064] In the context of amino acid sequences, a “sequence variant” is a modified sequence that has one or more amino acids deleted, substituted, or inserted compared to a reference amino acid sequence. As a result of the modification, such a sequence variant has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having 10 or fewer modifications, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence is “at least 90% identical” to the reference sequence.

[0065] A "conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding properties of a particular protein. Generally, a conservative substitution is one in which a substituted amino acid residue is replaced by an amino acid residue with a similar side chain. Conservative substitutions include those found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be classified into conserved substitution groups based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, aliphatic groups may include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other conserved substituents include sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negative-charged residues and their amides: Asp, Asn, Glu, and Gln; polar positive-charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0066] Amino acid insertions can include amino-terminus and / or carboxyl-terminus fusions extending from one residue to a polypeptide containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion of an amino acid sequence to the N-terminus or C-terminus of a reporter molecule or enzyme.

[0067] In general, modifications in sequence variants do not invalidate or significantly reduce the desired functionality of the respective reference sequence. For example, it is preferable that the variant sequences of this disclosure do not significantly reduce or completely invalidate the functionality of the sequence of an antibody or antigen-binding fragment to bind to the same epitope, compared to an antibody or antigen-binding fragment having (or encoding) the reference sequence. Guidance for determining which nucleotides and amino acid residues can be substituted, inserted, or deleted without invalidating the desired structure or function can be found, for example, by using a known computer program.

[0068] As used herein, a nucleic acid sequence or amino acid sequence “derived” from a specified nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein. An amino acid sequence derived from a nucleic acid sequence or a particular sequence may have an amino acid sequence that is essentially identical to the sequence or a portion thereof from which it originates, and thus “essentially identical” includes the sequence variants as defined above. An amino acid sequence derived from a nucleic acid sequence or a particular peptide or protein may originate from a corresponding domain in a particular peptide or protein. In this context, “corresponding” means having the same functionality or properties for the purpose. For example, “extracellular domain” corresponds to another “extracellular domain” (of another protein), or “transmembrane domain” corresponds to another “transmembrane domain” (of another protein). Thus, “corresponding” portions of peptides, proteins, and nucleic acids are readily identifiable to those skilled in the art. Similarly, a sequence “derived” from another (e.g., “source”) sequence can be identified by those skilled in the art as having its origin in the source sequence.

[0069] A nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein from which it originates. However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations compared to the starting nucleic acid, peptide, polypeptide, or protein from which it originates, and in particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant of the starting nucleic acid, peptide, polypeptide, or protein from which it originates. For example, in a peptide / protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residues may be inserted or deleted.

[0070] As used herein, the term “mutation” refers to changes in nucleic acid sequences and / or amino acid sequences compared to a reference sequence, e.g., a corresponding genome, a wild type, or a reference sequence. Mutations may be, for example, somatic mutations (naturally occurring), spontaneous mutations, induced mutations (e.g., those induced by enzymes, chemicals, or radiation), or mutations obtained by site-directed mutagenesis (a molecular biological method for making specific and intentional changes to nucleic acid sequences and / or amino acid sequences) compared to a reference genome sequence. Thus, the terms “mutation” or “mutating” should be understood to also include, for example, the physical production or induction of mutations in nucleic acid sequences or amino acid sequences. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding the amino acid sequence in order to express a (recombinant) mutant polypeptide. Mutations can be achieved, for example, by altering a codon in a nucleic acid molecule encoding one amino acid (for example, by changing one, two, or three nucleotide bases within it) (for example, by site-directed mutagenesis) to provide a codon encoding a different amino acid or the same amino acid, or by synthesizing a sequence variant.

[0071] In the context of inserting nucleic acid molecules into cells, the term “introduction” means “transfection,” “transformation,” or “transformation,” and includes references to the incorporation of nucleic acid molecules into eukaryotic or prokaryotic cells, whether the nucleic acid molecule can be integrated into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0072] As used herein, the term “recombinant” (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid, etc.) refers to any molecule (antibody, protein, nucleic acid, etc.) that is prepared, expressed, produced, or isolated by recombinant means and does not exist in nature. “Recombinant” can be used synonymously with “engineered” or “unnatural” and may refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has been modified by introducing at least one genetic alteration or an exogenous nucleic acid molecule, such alterations or modifications being introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, alterations that introduce an expressible nucleic acid molecule encoding a protein, fusion protein, or enzyme, or the addition, deletion or substitution of other nucleic acid molecules or other functional disruption of the cellular genetic material. Further modifications include, for example, non-coding regulatory regions in which the modification alters the expression of a polynucleotide, gene, or operon.

[0073] As used herein, “heterogeneous,” “non-endogenous,” or “exogenous” means any gene, protein, compound, nucleic acid molecule or activity that is not native to the host cell or subject, or any gene, protein, compound, nucleic acid molecule or activity that is native to the host cell or subject and has been modified. Heterogeneous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that they differ in structure, activity, or both from the native gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterogeneous, non-endogenous, or exogenous gene, protein, or nucleic acid molecule may not be endogenous to the host cell or subject, and instead, the nucleic acid encoding such a gene, protein, or nucleic acid molecule may be added to the host cell by conjugation, transformation, transfection, electroporation, etc., and the added nucleic acid molecule may be incorporated into the host cell genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The terms "homologous" or "homogenetic" refer to genes, proteins, compounds, nucleic acid molecules, or activities found in or derived from a host cell, species, or strain. For example, a heterogeneous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and may encode homologous polypeptides or activities, but the polynucleotide or polypeptide may have altered structures, sequences, expression levels, or any combination thereof. Non-endogenous polynucleotides or genes, as well as the encoded polypeptides or activities, may originate from the same species, different species, or combinations thereof.

[0074] As used herein, the terms “endogenous” or “natural” refer to polynucleotides, genes, proteins, compounds, molecules, or activities that are normally present in a host cell or subject.

[0075] Where used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable, and all such designations include offspring. Therefore, the terms “transformed” and “transformed cell” include primary target cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that all offspring may not have exactly the same DNA content due to intentional or accidental mutations. Variant offspring with the same or substantially the same function, phenotype, or biological activity as those screened in the initially transformed cells are included. Where a clear designation is intended, it will be evident from the context.

[0076] As used herein, the terms “isolated” or “partially purified” refer to a nucleic acid, polypeptide, or protein isolated from at least one other component (e.g., nucleic acid, polypeptide, or protein) that is present with the nucleic acid, polypeptide, or protein found in its natural source and / or present with the nucleic acid, polypeptide, or protein when expressed by a cell, or, in the case of secreted polypeptides and proteins, would be secreted. A nucleic acid, polypeptide, or protein synthesized chemically, or synthesized using in vitro transcription / translation, is considered “isolated.” The terms “purified” or “substantially purified” refer to an isolated nucleic acid, polypeptide, or protein that is at least 95% by weight of the nucleic acid, polypeptide, or protein in question (e.g., containing or exceeding at least 96%, at least 97%, at least 98%, at least 99%).

[0077] CD8 alpha (or CD8a) is a protein expressed on T cells, including regulatory T cells. CD8 alpha polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_001759.3, NP001139345.1, NP_741969.1, NP_001369627.1, NP_757362.1, NP_001171571.1, NP_742100.1, NP_742099.1, and NP_004922, and these sequences are incorporated herein by reference.

[0078] KIR (killer immunoglobulin-like receptor) proteins are cell surface molecules expressed on natural killer (NK) cells and some T cells. The KIR gene family includes several loci associated with different protein structures having two or three domains and short or long cytoplasmic tails. Some KIR receptors are inhibitory, while others are activating, and one KIR receptor, KIR2DL4, can transmit both inhibitory and activating signals (see Debska-Zielkowska et al., Cells 10(7):1777, 2021).

[0079] KIR3DL1 is a protein expressed on NK cells and some T cells. It is also known as CD158E1, KIR, KIR2DL5B, KIR3DL1 / S1, NKAT-3, NKAT3, NKB1, and NKB1B. Examples of KIR3DL1 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_037421.2 and NP_001309097.1, and these sequences are incorporated herein by reference.

[0080] KIR3DL2 is a protein expressed on NK cells and some T cells. It is also known as 3DL2, CD158K, KIR-3DL2, NKAT-4, NKAT4, NKAT4B, and p140. Examples of KIR3DL2 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_006728.2 and NP_001229796.1, which are incorporated herein by reference.

[0081] KIR2DL1 is a protein expressed on NK cells and some T cells. It is also known as CD158A, KIR-K64, KIR221, KIR2DL3, NKAT, NKAT-1, NKAT1, and p58.1. Examples of KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_055033.2, which is incorporated herein by reference.

[0082] KIR2DL2 is a protein expressed on NK cells and some T cells. It is also known as CD158B1, CD158b, NKAT-6, NKAT6, and p58.2. Examples of KIR2DL2 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_055034.2, which is incorporated herein by reference.

[0083] KIR2DL3 is a protein expressed on NK cells and some T cells. It is also known as CD158B2, CD158b, GL183, KIR-023GB, KIR-K7b, KIR-K7c, KIR2DL, KIR2DS5, KIRCL23, NKAT, NKAT2, NKAT2A, NKAT2B, and p58. Examples of KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_056952.2, which is incorporated herein by reference.

[0084] II. Antibodies and Antigen-Binding Fragments In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that can bind to CD8. In some embodiments, such an antibody or antigen-binding fragment can bind to CD8+ regulatory T cells (Tregs) and modulate their activity. In some embodiments, the CD8+ Tregs are also KIR+.

[0085] Antibodies generally consist of a heavy chain and a light chain. Each heavy chain consists of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may contain three domains CH1, CH2, and CH3, and optionally a fourth domain CH4. Each of these domains is referred to as an "Fc domain." As used herein, if a binder contains an Fc domain, it may contain one or more Fc domains or entire Fc regions unless otherwise specified by the context. Each light chain consists of a variable region (abbreviated as VL) and a constant region or constant domain. The light chain constant region is the CL domain. The VH and VL regions are further divided into a hypervariable region called the complementarity-determining region (CDR), which may contain a conserved region called the framework region (FR) between them. Therefore, each VH and VL region consists of three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art.

[0086] As used herein, unless the context explicitly indicates otherwise, “antibody” refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (however, it is understood that heavy-chain antibodies lacking light chains are still included in the term “antibody”), as well as any antigen-binding portion or fragment of an intact antibody having or retaining the ability to bind to an antigen-targeting molecule recognized by the intact antibody, e.g., scFv, Fab, or F(ab')2 fragment. Thus, the term “antibody” as used herein is used in its broadest sense and includes intact antibodies comprising fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments comprising single-chain variable fragments (scFv), and polyclonal and monoclonal antibodies comprising their functional (antigen-binding) antibody fragments. This term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, e.g., intrabodies, peptidebodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies; multispecificity, e.g., bispecificity, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term “antibody” should be understood to include its functional antibody fragment. This term also encompasses intact or full-length antibodies, including antibodies of any class or subclass of IgG and its subclasses, including IgM, IgE, IgA, and IgD.

[0087] As used herein, the terms “antigen-binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment of the antibody of this disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

[0088] Human antibodies are well known (e.g., van Dijk and van de Winkel, Curr. Opin. Chem. Biol. 5:368-374, 2001). Human antibodies can be produced in transgenic animals (e.g., mice) that, upon immunization, can produce a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transplantation of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge (e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-2555, 1993; Jakobovits et al., Nature 362:255-258, 1993; Bruggemann et al., Year Immunol. 7:3340, 1993). Human antibodies can also be produced using phage display libraries (Hoogenboom and Winter, J.Mol.Biol.227:381-388, 1992; Marks et al., J.Mol.Biol.222:581-597, 1991). The techniques of Cole et al. and Boerner et al. are also applicable to the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77(1985); and Boerner et al., J.Immunol.147:86-95, 1991). Human monoclonal antibodies can be prepared by using improved EBV-B cell immortalization, as described by Traggiai et al. (Nat Med.10(8):871-875, 2004). As used herein, the term “human antibody” also includes such antibodies modified, for example, in a variable region, to produce the properties of the antibodies and antibody fragments of this disclosure.

[0089] The antibodies provided herein may be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD, i.e., including α, γ, μ, ε, or δ heavy chains). Within the IgG isotype, for example, the antibody may be an IgG1, IgG2, IgG3, or IgG4 subclass. In a specific embodiment, the antibody provided herein is an IgG1 antibody. The antibodies or antigen-binding fragments provided herein may include κ or λ light chains.

[0090] As used herein, the term “variable region” (light chain variable region (VL), heavy chain variable region (VH)) most often refers to each variable region polypeptide of the light and heavy chain pairs that are directly involved in the binding of the antibody to the antigen. The terms “VL” and “VH” refer to the variable binding regions derived from the light and heavy chains of the antibody, respectively. The variable binding region consists of distinct, well-defined subregions known as “complementarity-determining regions” (CDRs) and “framework regions” (FRs). The terms “complementarity-determining regions” and “CDRs” are synonymous with “hypervariable regions” or “HVRs,” and are known in the art to refer to sequences of amino acids within the TCR or antibody variable region that confer antigen specificity and / or binding affinity and are separated by framework sequences. Generally, each variable region of an immunoglobulin-binding protein has three CDRs. For example, in the case of an antibody, the VH and VL regions contain six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively). As used herein, a “variant” of a CDR refers to a functional variant of a CDR sequence having up to one to three amino acid substitutions, deletions, or combinations thereof.

[0091] In certain embodiments, it will be understood that the antibody or antigen-binding fragments of the Disclosure may include all or part of a heavy chain (HC), a light chain (LC), or both. For example, a full-length intact IgG antibody monomer typically includes VH, CH1, CH2, CH3, VL, and CL. The Fc component is further described herein. In certain embodiments, the antibody or antigen-binding fragments of the Disclosure may each include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each consisting of either the VH sequence or the VL sequence of the Disclosure.

[0092] The antibody fragments described herein can be obtained from antibodies by methods including digestion with enzymes such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be obtained by cloning and expression of a portion of the heavy chain or light chain sequence. This disclosure includes, for example, scFv containing CDRs derived from the antibodies described herein, heavy chain or light chain monomers and dimers, single-domain heavy chain antibodies, single-domain light chain antibodies, and single-chain antibodies in which the heavy chain and light chain variable domains are linked by a peptide linker.

[0093] In certain embodiments, the antibody or antigen-binding fragment according to the Disclosure includes a purified antibody, a monoclonal antibody, a single-chain antibody, Fab, Fab', F(ab')2, Fv, or scFv.

[0094] Throughout this disclosure, antibodies, their antigen-binding fragments, and fusion proteins may be referred to individually or collectively (for example, in any combination) as “binding proteins.”

[0095] The binding proteins described herein may be provided in a purified form. For example, the antibody may be present in a composition substantially free of other polypeptides, such that less than 90% (by weight), typically less than 60%, and more typically less than 50%, of the composition consists of other polypeptides.

[0096] The binding proteins of this disclosure may be immunogenic in human and / or non-human (or heterologous) hosts, for example, in mice. For example, an antibody may have an idiotope that is immunogenic in a non-human host but not immunogenic in a human host. Antibodies of this disclosure for human use typically include those not isolated from hosts such as mice, goats, rabbits, rats, and non-primate mammals, and in some cases not obtained by humanization or from heterologous mice. Variant forms of the disclosed antibodies that are manipulated to mitigate known or potential immunogenicity and / or other potential risks, or to confer desired structure and / or functionality to the antibody in non-human animals such as mice, are also contemplated herein (e.g., “moolyzed” antibodies for model studies using mice, for example, in mice, where one or more human amino acid residues, sequences, or motifs are replaced with residues, sequences, or motifs that have reduced or suppressed immunogenicity or other liabilities, or that have a desired structure and / or function).

[0097] Antibodies or antigen-binding fragments, such as those described herein, including but not limited to scFv, may, in certain embodiments, be contained in a fusion protein capable of specifically binding to an antigen described herein. As used herein, “fusion protein” means a protein having at least two distinct domains or motifs in a single strand, the domains or motifs not found together or in a given arrangement in nature within the protein. Polynucleotides encoding a fusion protein can be constructed using PCR, recombination, etc., or such fusion proteins can be synthesized.

[0098] Immunoglobulin sequences can be numbered according to numbering schemes (e.g., Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo), which allows annotation of equivalent residue positions and comparison of different molecules using the Antibody Numbering And Receptor Classification (ANARCI) software tool (see also Bioinformatics 15:298-300, 2016; Dondelinger et al., Front. Immunol. 9:2278, 2018).

[0099] As used herein, "specifically binds" or "specific for" refers to an affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M) of a binding protein (e.g., an antibody or an antigen-binding fragment thereof) or binding domain for a target molecule that is equal to or greater than 10 5 M -1 such that the binding (union) occurs at an association or binding with a rate of association [K on and a rate of dissociation [K off for this association reaction, and does not significantly associate or bind with any other molecule or component in the sample. The binding protein or binding domain can be classified as a "high-affinity" binding protein or binding domain or a "low-affinity" binding protein or binding domain. A "high-affinity" binding protein or binding domain has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 2]]M -1 , at least 10 12 M -1 , or at least 10 13 M -1This refers to the binding protein or binding domain that it possesses. A "low affinity" binding protein or binding domain can have up to 10 7 M -1 , up to 10 6 M -1 , or up to 10 5 M -1 This refers to a binding protein or binding domain having a Ka of 10. Alternatively, affinity is expressed in M ​​units (e.g., 10). -5 M~10 -13 It can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction in M). The terms “binding” and “specific binding” and similar references do not include non-specific sticking.

[0100] The binding of binding proteins can be determined or evaluated using appropriate assays such as surface plasmon resonance (SPR), e.g., the Biacore® system; kinetic exclusion assays such as KinExA®; and biolayer interferometry (e.g., using the ForteBio® Octet platform); isothermal titration calorimetry (ITC), e.g., antigen-binding ELISA (e.g., direct or indirect) using optical density imaging at 450 nm or flow cytometry imaging.

[0101] The term "epitope" or "antigen epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a corresponding binding molecule such as an immunoglobulin or other binding molecule, domain, or protein. Epitope determinants generally include chemically active surface groups of molecules such as amino acids or sugar side chains, and may have specific three-dimensional structural properties as well as specific charge properties. Epitopes to which binding proteins bind can be linear (continuous) or 3D (discontinuous). Linear or continuous epitopes are epitopes recognized by antibodies according to the linear sequence of amino acids or primary structure. 3D epitopes can be recognized according to their three-dimensional shape and protein structure. In the case of 3D epitopes (3D structures), since amino acid sequences typically form a 3D structure as epitopes, the amino acids forming the epitope may or may not be adjacent to the primary structure (i.e., they may or may not be consecutive amino acids in the amino acid sequence).

[0102] Multispecific antibodies and binding proteins The antibodies and antigen-binding fragments of this disclosure may, in embodiments, be multispecific (e.g., bispecific, triplicate, quadruplicate, etc.) and may be provided in any multispecific format disclosed herein. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites or antigens. In certain embodiments, the antibody or antigen-binding fragment of this disclosure is a multispecific antibody, such as a bispecific or triplicate antibody. The formats of bispecific antibodies are disclosed, for example, in Spiess et al. (Mol.Immunol. 67(2):95, 2015) and Brinkmann and Kontermann (mAbs 9(2):182-212, 2017), and these bispecific formats and methods for their preparation are incorporated herein by reference, for example, bispecific T cell engagers (BiTE), DART, knob-into-hole (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, TandAbs, triple bodies, TriBi minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, intrabodies, CrossMab, dual-acting Fab (DAF) (two-in-one or four-in-one), DutaMabs, DT-IgG, charge pair (Charge Examples include pairs, Fab arm replacement, SEED body, Triomabs, LUZ-Y assembly, Fcab, κλ body, orthogonal Fab, DVD-IgGs, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one).Examples of bispecific and multispecific antibodies include scFv1-ScFv2, ScFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-Fc, dAb-IgG, IgG-VHH, Tandem scFv-Fc, (scFv1)2-Fc-(VHH)2, scFv-Fc, one-arm tandem scFv-Fc, and DART-Fc. IgG-scFv can be IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv.

[0103] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537, 1983; International Publication No. 93 / 08829; Traunecker et al., EMBO J.10:3655, 1991) and the "knob-in-hole" operation (e.g., U.S. Patent Nos. 5,731,168). Multispecific antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimer molecules (International Publication No. 2009 / 089004A1); crosslinking two or more antibodies or fragments (e.g., U.S. Patent No. 4,676,980; Brennan et al., Science 229:81, 1985); producing bispecific antibodies using leucine zippers (e.g., Kostelny et al., J.Immunol. 148(5):1547-1553, 1992); or using "diabody" techniques to produce bispecific antibody fragments (e.g., Hollinger et al., Proc.Natl.Acad.Sci.USA). They can be prepared by using single-chain Fv(scFv) dimers (see, for example, Gruber et al., J.Immunol. 152:5368, 1994); and by preparing triplicate antibodies (as described, for example, Tutt et al., J.Immunol. 147:60, 1991).

[0104] Manipulated antibodies having three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0105] The antibodies or antigen-binding fragments disclosed herein also include “dual-action Fabs” or “DAFs” that include antigen-binding sites that bind to two different antigens (e.g., U.S. Patent Application Publication No. 2008 / 0069820, Bostrom et al., Science 323:1610-1614, 2009).

[0106] The "CrossMab" antibody is also included herein (see, for example, International Publication Nos. 2009 / 080251, 2009 / 080252; 2009 / 080253; 2009 / 080254; and 2013 / 026833).

[0107] In some embodiments, the antibody or antigen-binding fragments disclosed herein contain different antigen-binding sites fused to one or the other of two subunits of the Fc domain. Thus, the two subunits of the Fc domain may constitute two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two polypeptides. To improve the yield and purity of bispecific molecules in recombinant production, it is advantageous to introduce modifications to the Fc domain of the binder that facilitate the association of the desired polypeptides.

[0108] Accordingly, certain embodiments relate to a binder (e.g., an antibody or its antigen-binding fragment) comprising an Fc domain consisting of (a) at least a first binding domain, (b) a second binding domain, and (c) first and second subunits capable of stable association, wherein the Fc domain includes modifications that facilitate the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Accordingly, in one embodiment, the modifications are located within the CH3 domain of the Fc domain.

[0109] In certain embodiments, the Fc modification is a so-called "knob-into-hole" modification, where one of the two subunits of the Fc domain contains a "knob" modification and the other of the two subunits of the Fc domain contains a "hole" modification. In certain embodiments, the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index). Knob-into-hole techniques are described, for example, in U.S. Patents 5,731,168 and 7,695,936, and by Ridgway et al. (Prot Eng 9:617-621, 1996) and Carter (J Immunol Meth 248:7-15, 2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the protrusion may be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The protrusion is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller side chain (e.g., alanine or threonine).

[0110] Accordingly, in some embodiments, in the CH3 domain of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a projection within the CH3 domain that can be positioned in a cavity within the CH3 domain of the second Fc domain, and in the CH3 domain of the second Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second Fc domain that can be positioned in the projection within the CH3 domain of the first Fc domain. The projection and cavity can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In certain embodiments, in the CH3 domain of the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In another embodiment, in the second Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0111] In some embodiments, the first Fc domain is further modified by replacing the serine residue at position 354 with a cysteine ​​residue (S354C), and the second Fc domain is further modified by replacing the tyrosine residue at position 349 with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two Fc domains, which further stabilizes the dimer (Carter, J Immunol Methods 248:7-15, 2009). In some embodiments, the first Fc domain includes amino acid substitutions S354C and T366W (EU numbering), and the second Fc domain includes amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0112] In some embodiments, modifications that facilitate the association of the first and second Fc domains include modifications that mediate an electrostatic steering effect, as described, for example, in PCT International Publication 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domains with charged amino acid residues such that homodimerization is electrostatically unfavorable, but heterodimerization is electrostatically favorable.

[0113] In some embodiments, the binder (e.g., an antibody or its antigen-binding fragment) comprises one or more scFv or “single-chain variable fragments.” An scFv is a fusion protein of the heavy-chain (VH) variable region and the light-chain (VL) variable region of an antibody, linked by a short linker peptide of 10 to about 25 amino acids. The linker is typically glycine-rich for flexibility and serine or threonine-rich for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. ScFv antibodies are described, for example, by Houston (Methods in Enzymol. 203:46-96, 1991). Methods for constructing scFv molecules and designing appropriate peptide linkers are described, for example, in U.S. Patents 4,704,692 and 4,946,778; Raag and Whitlow (FASEB 9:73-80, 1995); and Bird and Walker (TIBTECH 9:132-137, 1991).

[0114] Binding agents that are scFv-Fc (e.g., antibodies or their antigen-binding fragments) have been described by Sokolowska-Wedzina et al. (Mol. Cancer Res. 15(8):1040-1050, 2017).

[0115] In some embodiments, the binder (e.g., an antibody or its antigen-binding fragment) is a “bispecific T cell engager” or BiTE (see, e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261, and 2008 / 119567). This approach utilizes two antibody variable domains located on a single polypeptide. For example, a single polypeptide chain may contain two single-chain Fv(scFv) fragments, each having variable heavy (VH) and variable light (VL) domains separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific to different proteins so that both proteins are bound by the BiTE. Since it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, such as the CHO cell line. However, specific purification techniques (see, for example, European Patent No. 1691833) may be required to separate the monomer's bispecific T cell engager from other multimer species that may have biological activities other than the monomer's intended activity. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography to elute the polypeptide using an imidazole concentration gradient. This eluate is further purified using anion exchange chromatography to elute the polypeptide using a sodium chloride concentration gradient. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimer species. In some embodiments, the conjugate, which is a bispecific antibody, consists of a single polypeptide chain containing two single-stranded FV fragments (scFVs) fused to each other by a peptide linker.

[0116] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies may originate from the variable domain of an antibody heavy chain derived from a camelid (e.g., a nanobody or VHH fragment). Furthermore, the term single-domain antibody includes autonomous human heavy chain variable domains (aVH) or VNAR fragments derived from sharks (see, e.g., Hasler et al., Mol.Immunol.75:28-37, 2016). Techniques for producing single-domain antibodies (DABs or VHHs) are well known in the art, as disclosed, for example, by Cossins et al. (Prot Express Purif 51:253-259, 2006) and Li et al. (Immunol.Lett.188:89-95, 2017). Single-domain antibodies can be obtained from camels, alpacas, or llamas by standard immunization techniques, for example (see Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007). VHH can have potent antigen-binding ability and can interact with novel epitopes inaccessible to conventional VH-VL pairs (see Muyldermans et al., 2001 above). Alpaca serum IgG contains approximately 50% heavy-chain-only IgG antibodies (HCAbs) from camelids (see Maass et al., 2007 above). Alpacas can be immunized with antigens, and VHH that binds to and neutralizes target antigens can be isolated (see Maass et al., 2007 above). PCR primers for amplifying the alpaca VHH coding sequence have been identified and can be used to construct an alpaca VHH phage display library, which can then be used for isolation of antibody fragments by standard biopanning techniques well known in the art (see, for example, Maass et al., 2007 above).

[0117] In some embodiments, the binder (e.g., an antibody or its antigen-binding fragment) is IgG-scFV. Examples of IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody formats and methods for their preparation are described, for example, by Brinkmann and Kontermann (MAbs 9(2):182-212, 2017); Wang et al. (Antibodies 8:43, 2019); Dong et al. (MAbs 3:273-88, 2011); Natsume et al. (J. Biochem. 140(3):359-368, 2006); Cheal et al. (Mol. Cancer Ther. 13(7):1803-1812, 2014); and Bates and Power (Antibodies 8:28, 2019).

[0118] Igg-like bivariable domain antibodies (DVD-Ig) have been described by Wu et al. (Nat Biotechnol 25:1290-97, 2007), by Hasler et al. (Mol.Immunol. 75:28-37, 2016), and in International Publication Nos. 08 / 024188 and 07 / 024715.

[0119] Triomab was described by Chelius et al. (MAbs 2(3):309-319, 2010). 2-in-1-IgG was described by Kontermann et al. (Drug Discovery Today 20(7):838-847, 2015).

[0120] Tandem antibodies, or TandAb, were described by Kontermann et al. (Drug Discovery Today 20(7):838-847, 2015).

[0121] The ScFv-HSA-scFv antibody has also been described by Kontermann et al. (Drug Discovery Today 20(7):838-847, 2015).

[0122] In some embodiments, the binder (e.g., an antibody or its antigen-binding fragment) is a scaffold antigen-binding protein, such as fibronectin and designed ankyrin repeat proteins (DARPins), used as an alternative scaffold for the antigen-binding domain (see, e.g., Gebauer and Skerra Curr Opin Chem Biol 13:245-255, 2009; Stumpp et al., Drug Discovery Today 13:695-701, 2008). In some embodiments, the scaffold antigen-binding protein is selected from the group consisting of lipocalin (Anticalin), protein A-derived molecules such as Z domains (Affibody) derived from protein A, A domains (Avimer / Maxibody), serum transferrin (trans-body), designed ankyrin repeat protein (DARPin), fibronectin (AdNectin), C-type lectin domain (Tetranectin), beta-lactamase (VNAR fragment), which is a variable domain of a novel antigen receptor, human gamma crystallin or ubiquitin (Affilin molecule), Knitz-type domain of human protease inhibitors, and proteins of the knottin family, peptide aptamers, and microbodies such as fibronectin (adnectin). Lipocalin is a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilines, retinoids, and lipids. They have a rigid beta-sheet secondary structure with several loops at the open end of a conical structure that can be manipulated to bind to different target antigens. Antikarin is 160-180 amino acids in size and is derived from lipocalin. (For further details, see Biochim Biophys Acta 1482:337-350,2000, U.S. Patent No. 7,250,297B1; U.S. Patent Application Publication No. 20070224633)

[0123] Designer ankyrin repeat proteins (DARPins) are derived from ankyrin, a family of proteins that mediate the attachment of endogenous membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and beta-turns. They can be engineered to bind to different target antigens by randomizing the residues in the first alpha-helices and beta-turns of each repeat. Their binding interfaces can be increased by increasing the number of modules (affinity maturation method). (For further details, see J.Mol.Biol.332:489-503,2003;PNAS 100(4):1700-1705,2003;J.Mol.Biol.3691015-1028;2007;US Patent Application Publication No. 20040132028A1).

[0124] Fc domain modification In some embodiments, the binding protein of this disclosure (e.g., an antibody or its antigen-binding fragment) includes an Fc moiety. In certain embodiments, the Fc moiety may be of human origin, e.g., human IgG1, IgG2, IgG3 and / or IgG4, or may be of another Ig class or isotype. In certain embodiments, the antibody or antigen-binding fragment may include an Fc moiety derived from human IgG1.

[0125] As used herein, the term “Fc moiety” refers to a sequence that includes, consists of, essentially consists of, or derives from, a portion of an immunoglobulin heavy chain, beginning in the hinge region immediately upstream of a papain cleavage site (e.g., residue 216 in natural IgG, with the first residue of the heavy chain constant region being 114) and ending at the C-terminus of the immunoglobulin heavy chain. Thus, an Fc moiety may be a complete Fc moiety or a portion thereof (e.g., a domain). In certain embodiments, a complete Fc moiety includes a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216–446). Further lysine residues (K) may be present at the C-terminus of the Fc moiety but are often cleaved by mature antibodies. Amino acid positions within the Fc moiety can be numbered according to Kabat’s EU numbering system (see, for example, Kabat et al., “Sequences of Proteins of Immunological Interest”, USDept. Health and Human Services, 1983 and 1987). The amino acid positions in the Fc region can also be numbered according to the IMGT numbering system (including the unique numbering of the C domain and exon numbering) and the Kabat numbering system.

[0126] In some embodiments, the Fc portion includes at least one of the following: a hinge domain (e.g., upper hinge region, intermediate hinge region, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. In some embodiments, the Fc portion includes at least a hinge domain, a CH2 domain, or a CH3 domain. In further embodiments, the Fc portion is a complete Fc portion. The Fc portion may also include one or more amino acid insertions, deletions, or substitutions compared to naturally occurring Fc portions. For example, at least one of the hinge domain, the CH2 domain, or the CH3 domain, or a portion thereof, may be deleted. For example, the Fc portion may include, or consist of, (i) a hinge domain (or part thereof) fused to a CH2 domain (or part thereof), (ii) a hinge domain (or part thereof) fused to a CH3 domain (or part thereof), (iii) a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof), (iv) a hinge domain (or part thereof), (v) a CH2 domain (or part thereof), or (vi) a CH3 domain or part thereof.

[0127] The Fc moieties of this disclosure can be modified to alter the amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule while retaining or enhancing at least one desirable function conferred by the naturally occurring Fc moiety, and / or reducing an undesirable function of the naturally occurring Fc moiety. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. The portions of naturally occurring Fc moieties involved in such functions are described in the Art.

[0128] In some embodiments, the Fc region or Fc domain substantially does not bind to at least one Fc receptor selected from FcyRI(CD64), FcyRIIA(CD32a), FcyRIIB(CD32b), FcyRIIIA(CD16a), and FcyRIIIB(CD16b). In some embodiments, the Fc region or domain exhibits substantially no binding to any of the Fc receptors selected from FcyRI(CD64), FcyRIIA(CD32a), FcyRIIB(CD32b), FcyRIIIA(CD16a), and FcyRIIIB(CD16b). As used herein, “substantially no binding” means weak or no binding to one or more selected Fc gamma receptors. In some embodiments, “substantially no binding” means at least a 1000-fold decrease in binding affinity (e.g., increased Kd) to the Fc gamma receptor. In some embodiments, the Fc domain or region is Fc null. As used herein, “Fc null” refers to an Fc region or domain that has weak or no binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or region exhibits at least a 1000-fold decrease in binding affinity to the Fc gamma receptor (e.g., an increase in Kd).

[0129] In some embodiments, the Fc domain exhibits reduced or substantially absent effector functional activity. As used herein, “effector functional activity” refers to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP, or CDC activity compared to a wild-type Fc domain. In some embodiments, the Fc domain shows a decrease in ADCC, ADCP, and CDC compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits substantially no effector function (i.e., the ability to stimulate ADCC, ADCP, or CDC). As used herein, “substantially absent effector function” refers to a decrease in effector functional activity of at least 1000-fold compared to a wild-type Fc domain.

[0130] In some embodiments, the Fc domain has reduced or no ADCC activity. As used herein, reduced or no ADCC activity refers to a reduction in the ADCC activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0131] In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or no CDC activity refers to a reduction in the CDC activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0132] In vitro and / or in vivo cytotoxic assays can be performed to confirm reduced / depleted ADCC and / or CDC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks the Fcγ receptor (and therefore likely lacks ADCC activity). Primary cells and NK cells, which mediate ADCC, express only Fcgamma RIII, while monocytes express Fcgamma RI, Fcgamma RII, and Fcgamma RIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet (Annu. Rev. Immunol. 9:457-492, 1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063, 1986; Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502, 1985; U.S. Patent No. 5,821,337; Bruggemann et al., J. Exp. Med. 166:1351-1361, 1987). Alternatively, non-radioactive assay methods may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, in animal models, such as those disclosed by Clynes et al. (Proc. Nat'l Acad. Sci. USA 95:652-656, 1998).

[0133] C1q binding assays can also be performed to confirm that an antibody or Fc domain or region is unable to bind to C1q and therefore lacks or has reduced CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publications 2006 / 029879 and 2005 / 100402. CDC assays can also be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163, 1996; Cragg et al., Blood 101:1045-1052, 2003; Cragg and Glennie, Blood 103:2738-2743, 2004).

[0134] In some embodiments, the Fc domain has reduced or no ADCP activity. As used herein, reduced or no ADCP activity refers to a reduction in the ADCP activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0135] ADCP binding assays may also be performed to determine whether an antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity (see, for example, U.S. Patent Application Publication No. 20190079077 and U.S. Patent Application Publication No. 20190048078 and the references disclosed herein).

[0136] Antibodies exhibiting reduced effector activity include those with one or more substitutions among Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (see U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (see U.S. Patent No. 7,332,581), as well as Fc variants with two or more substitutions among amino acid positions 265, 269, 270, 297, and 327. Certain antibody variants with reduced binding to FcR are also known (e.g., U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; Shields et al., J. Biol. Chem. 9(2):6591-6604, 2001).

[0137] In certain embodiments, the binder comprises an Fc domain or Fc region having one or more amino acid substitutions that reduce the Fc gamma-R bond, e.g., substitutions at positions 234 and 235 of the Fc region (residue EU numbering). In some embodiments, the substitutions are L234A and L235A (LALA). In some embodiments, the Fc domain further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region (see, e.g., International Publication No. 2012 / 130831). In some embodiments, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0138] In some embodiments, modifications are made to the Fc region that result in alterations (i.e., reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. (J.Immunol.164:4178-4184, 2000).

[0139] Production of binding proteins In various embodiments, binding proteins (e.g., antibodies or their antigen-binding fragments) can be produced in human, mouse, or other animal-derived cell lines. Binding agents can be produced using recombinant DNA expression. This allows for the production of antibodies in the optimal host species, as well as various antigen-binding moieties and other binding agents (including fusion proteins). The production of antibodies, their antigen-binding moieties, and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs is also an alternative to cell-based production systems. A major advantage of transgenic animals is the potential high yield from renewable resources.

[0140] Nucleic acid molecules encoding the amino acid sequence of an antibody or its antigen-binding moiety, as well as other binders, can be prepared by various methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding antibodies, antigen-binding moieties, or other binders. Furthermore, nucleotide sequences encoding antibodies or antigen-binding moieties and other binders can be prepared using oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis. As described herein, at least an antibody, its antigen-binding moiety, a binder, or a nucleic acid sequence encoding its polypeptide can be recombined with vector DNA according to the prior art, e.g., blunt-ended or staggered-ended ends for ligation, restriction enzyme digestion to provide suitable ends, sticky end packing as needed, alkaline phosphatase treatment to avoid undesirable ligation, and ligation with a suitable ligase. Techniques for such operations have been disclosed, for example, by Maniatis et al. (Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989)) and Ausubel et al. (Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993) and can be used to construct nucleic acid sequences and vectors encoding an antibody or its antigen-binding moiety or its VH and / or VL polypeptide. When the binder contains an antibody or its antigen-binding moiety, in some embodiments the VH polypeptide is encoded by a first nucleic acid. In some embodiments the VL polypeptide is encoded by a second nucleic acid. In some embodiments the VH and VL polypeptides are encoded by a single nucleic acid.

[0141] Nucleic acid molecules such as DNA are said to be "expressible" with polypeptides if they contain nucleotide sequences that contain transcriptional and translational regulatory information, and such sequences are "operably ligated" to nucleotide sequences that encode polypeptides. An operable ligation is a ligation that connects a regulatory DNA sequence to a DNA sequence that is to be expressed (e.g., an antibody or its antigen-binding portion) in such a way that it enables the gene expression of a recoverable amount of polypeptide(s) or antigen-binding portion. The exact nature of the regulatory region required for gene expression can vary from organism to organism, as is well known in similar fields (see, for example, Sambrook et al., 1989; Ausubel et al., 1987–1993).

[0142] Therefore, the expression of the antibodies or their antigen-binding moieties or other conjugates described herein may occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, such as yeast, insect, fungal, avian, and mammalian cells (either in vivo or in situ), or host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cells may be used. Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using a yeast ubiquitin hydrolase system. The fusion proteins thus produced may be processed in vivo or purified and processed in vitro, enabling the synthesis of the antibodies or their antigen-binding moieties described herein having specific amino-terminal sequences. Furthermore, problems associated with the retention of methionine residues derived from start codons in direct yeast (or bacterial) expression can be avoided (e.g., Sabin et al., 7 Bio / Technol. 705, 1989; Miller et al., 7 Bio / Technol. 698, 1989). Recombinant antibodies or their antigen-binding moieties or other binders can be obtained using one of a series of yeast gene expression systems that incorporate promoters and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, the promoter and terminator signals of phosphoglycerate kinase genes can be utilized.

[0143] The production of antibodies or their antigen-binding moieties and other binders in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express a polypeptide, using methods known to those skilled in the art. See Ausubel et al., 1987–1993.

[0144] In some embodiments, the introduced nucleic acid sequence (encoding an antibody or its antigen-binding portion or its polypeptide or other binder) is incorporated into a plasmid or viral vector capable of autonomous replication in recipient host cells. A wide variety of vectors can be used for this purpose and are known and available to those skilled in the art (see, for example, Ausubel et al., 1987–1993 above). Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells without the vector, the desired copy number of the vector within a particular host, and whether it is desirable that the vector can be "shuttled" between different species of host cells.

[0145] Examples of prokaryotic vectors known in the art include plasmids that can be replicated in Escherichia coli (E. coli). Other gene expression elements useful for expressing DNA encoding antibodies or their antigen-binding portions and other binders include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280, 1983), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777, 1982), and Moloney mouse leukemia virus LTR (Grosschedl et al., 41 Cell 885, 1985); (b) splice regions and polyadenylation sites, such as those derived from the SV40 late region (Okayama et al., 1983, see above); and (c) polyadenylation sites such as SV40 (Okayama et al., 1983, see above). The DNA genes encoding immunoglobulins may be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S globin intercalated sequence, the immunoglobulin and rabbit S globin polyadenylation sites, and the SV40 polyadenylation element as expression elements, as described by Liu et al. and Weidle et al. (51 Gene 21, 1987).

[0146] In the case of nucleotide sequences encoding immunoglobulins, the transcription promoter could be, for example, human cytomegalovirus, and the promoter enhancer could be cytomegalovirus and mouse / human immunoglobulin.

[0147] In some embodiments, for the expression of DNA coding regions in rodent cells, the transcription promoter may be a viral LTR sequence, and the transcription promoter enhancer may be either or both a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the expression elements listed above to achieve protein expression in mammalian cells.

[0148] Each coding region or gene fusion is assembled or inserted into an expression vector. Recipient cells capable of expressing the variable region(s) or its antigen-binding moiety are then transfected either alone with an antibody or antibody polypeptide or a nucleotide encoding its antigen-binding moiety, or co-transfected with a polynucleotide(s) encoding the VH and VL chain coding regions. The transfected recipient cells are cultured under conditions that allow expression of the incorporated coding region, and the expressed antibody chain or intact antibody or antigen-binding moiety is recovered from the culture.

[0149] In some embodiments, nucleic acids containing a coding region encoding an antibody or its antigen-binding portion are then assembled into a separate expression vector used to co-transfect recipient host cells. Each vector may contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, with the first selectable gene designed for selection in a bacterial system and the second selectable gene designed for selection in a eukaryotic system, and each vector having a set of coding regions. This strategy first results in a vector that directs the production of a nucleotide sequence in a bacterial system and enables amplification. The DNA vector thus produced and amplified in the bacterial host is then used to co-transfect eukaryotic cells, enabling the selection of co-transfected cells having the desired transfected nucleic acid (e.g., encoding the heavy and light chains of an antibody). Non-limiting examples of selectable genes for use in a bacterial system are genes that confer resistance to ampicillin and genes that confer resistance to chloramphenicol. Selectable genes for use as eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (named gpt) and the Tn5-derived phosphotransferase gene (named neo). Alternatively, fusion nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0150] For transfection of expression vectors and production of antibodies or their antigen-binding moieties or other binders, the recipient cell line may be a Chinese hamster ovary cell line (e.g., DG44) or myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin gene and have a mechanism for immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are recombinant Ig-producing myeloma cells SP2 / 0. SP2 / 0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, and the secreted immunoglobulins can be obtained from ascites fluid.

[0151] Expression vectors encoding antibodies or their antigen-binding moieties or other binders can be introduced into suitable host cells by any of a variety of suitable means, including, as is known to those skilled in the art, biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical means such as electroporation, direct microinjection, and microprojectile bombardment (Johnston et al., Science, 240:1538, 1988).

[0152] Yeast offers certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes polypeptides containing the leader sequence (i.e., pre-polypeptides) (see, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982)).

[0153] Yeast gene expression systems can routinely evaluate the production, secretion, and stability levels of antibodies, their aggregated antibodies, and antigen-binding moieties. Various yeast gene expression systems can be utilized, incorporating promoters and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich media. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translation elongation factor 1α promoter. Several approaches can be taken to evaluate the optimal expression plasmid for immunoglobulin expression in yeast (see, e.g., II DNA Cloning 45, (Glover, ed., IRL Press, 1985); U.S. Patent Application Publication No. 2006 / 0270045A1).

[0154] Bacterial strains can also be used as hosts for the production of antibody molecules or their antigen-binding moieties or other binders described herein. E. coli strains such as E. coli W3110 (K12 strain), Bacillus species, Enterobacteria such as Salmonella typhimurium and Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicons and regulatory sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vectors have replication sites and specific genes that can provide phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of antibodies and their antigen-binding moieties in bacteria (see Glover, 1985, above; Ausubel, 1987, 1993, above; Sambrook, 1989; Colligan, 1992–1996).

[0155] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications of immunoglobulin molecules, including leader peptide removal, VH and VL chain folding and assembly, antibody molecule glycosylation, and secretion of functional antibodies and / or their antigen-binding moieties.

[0156] In addition to the lymphoid cells mentioned above, fibroblast-derived cells such as Vero or CHO-K1 cells are mammalian cells that may be useful as hosts for antibody protein production. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-K1, and DG44 cells; PERC6® cells (Crucell); and NSO cells. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications to the heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced by 293 cells.

[0157] In some embodiments, one or more antibodies or their antigen-binding moieties or other binders may be produced in vivo in animals manipulated or transfected with one or more nucleic acid molecules encoding polypeptides, according to any suitable method.

[0158] In some embodiments, the antibody or its antigen-binding moiety is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, by Sitaraman et al. (Methods Mol. Biol. 498:229-244, 2009); Spirin (Trends Biotechnol. 22:538-545, 2004); and Endo et al. (Biotechnol. Adv. 21:695-713, 2003).

[0159] Many vector systems are available for the expression of VH and VL chains in mammalian cells (see Glover, 1985, above). Various approaches can be followed to obtain intact antibodies. As mentioned above, it is possible to achieve intracellular association and linkage of the VH and VL chains to the complete tetrameric H2L2 antibody or its antigen-binding moiety by co-expressing the VH and VL chains, and optionally the associated constant region, in the same cell. Co-expression can occur by using either the same or different plasmids in the same host. The nucleic acids encoding the VH and VL chains or their antigen-binding moieties can be placed in the same plasmid, which can then be transfected into cells to directly select cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the VL chain, and then the resulting cell line can be transfected with a VH chain plasmid containing a second selection marker. Cell lines that produce antibodies, their antigen-binding moieties, or other binders via any of these pathways can be transfected with plasmids encoding further copies of peptides, VH, VL, or VH+VL chains, along with additional selection markers, to create cell lines with enhanced properties, such as higher production of aggregated antibodies or their antigen-binding moieties, or enhanced stability of the transfected cell line.

[0160] Furthermore, plants are emerging as a convenient, safe, and economical alternative expression system for recombinant antibody production based on large-scale cultures of microbial or animal cells. Antibodies or antigen-binding moieties can be expressed in plant cell cultures or plants grown by conventional methods. Expression in plants may be systemic, limited to intracellular plastids, or limited to seeds (endosperm) (e.g., U.S. Publication No. 2003 / 0167531; U.S. Patent No. 6,080,560; U.S. Patent No. 6,512,162; International Publication No. 0129242). Several plant-derived antibodies have reached advanced stages of development, including clinical trials (e.g., Biolex, NC).

[0161] In the case of intact antibodies, the variable regions (VH and VL) of the antibody are typically ligated to the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells (see, for example, International Publication No. 87 / 02671, the entirety of which is incorporated herein by reference), according to well-known procedures. Antibodies may contain both light chain and heavy chain constant regions. The heavy chain constant region may contain CH1, hinge, CH2, CH3, and sometimes CH4 regions. In some embodiments, the CH2 domain may be deleted or omitted.

[0162] Alternatively, techniques described for the production of single-chain antibodies (see, for example, U.S. Patent No. 4,946,778; Bird, Science 242:423-42, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Ward et al., Nature 334:544-554, 1989, which are incorporated herein by reference in their entirety) can be adapted to produce single-chain antibodies that specifically bind to a desired antigen. Single-chain antibodies are formed by linking the heavy-chain variable region and the light-chain variable region of the Fv region via amino acid crosslinking, resulting in a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, for example, Skerra et al., Science 242:1038-1041, 1988, which are incorporated herein by reference in their entirety).

[0163] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or their antigen-binding moieties can be recovered and purified by known techniques, e.g., immunoadsorption or immunoaffinity chromatography, chromatography, e.g., HPLC (high-performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof (see Scopes, Protein Purification (Springer-Verlag, NY, 1982)). Substantially pure antibodies or their antigen-binding moieties with at least about 90%–95% homogeneity are advantageous, and antibodies or their antigen-binding moieties with 98%–99% or greater homogeneity are also particularly advantageous for pharmaceutical applications. Once partially or homogeneously purified as desired, the intact antibody or its antigen-binding moiety can subsequently be used therapeutically or in the development and implementation of assay procedures, immunofluorescence staining, etc. (see generally Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981)).

[0164] Anti-CD8 antibody and its antigen-binding fragment, and binding protein In some embodiments, the anti-CD8 antibody, its antigen-binding fragment, or binding protein of this disclosure comprises any of the substitutions in Table 1 or Table 2. In some embodiments, an anti-CD8 antibody, antigen-binding fragment, or binding protein comprising or consisting of one or more of SEQ ID NOs.17-60 is provided. In some embodiments, a binding protein having the sequence in Table 3 is provided.

[0165] III. Pharmaceutical Compositions or Formulations In some embodiments, the binders disclosed herein (e.g., antibodies and their antigen-binding fragments) relate to compositions comprising an active ingredient (i.e., a binder as described herein, or a nucleic acid encoding an antibody or its antigen-binding portion, or other binders as described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to an active agent combined with a pharmaceutically acceptable carrier approved for use in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio within the bounds of sound medical judgment.

[0166] The preparation of pharmacokinetic compositions containing an active ingredient dissolved or dispersed therein is well understood in the art and does not need to be limited based on a particular formulation. Typically, such compositions are prepared for injection as either a liquid solution or a suspension. However, solid forms suitable for rehydration in a liquid or suspension before use can also be prepared. Preparations may also be emulsified or presented as liposome compositions. Antibodies or their antigen-binding moieties or other binders may be pharmaceutically acceptable, compatible with the active ingredient, and can be mixed with excipients in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the pharmaceutical composition may contain small amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., that enhance or maintain the efficacy of the active ingredient (e.g., antibody or its antigen-binding moiety or other binder). The pharmaceutical compositions described herein may contain pharmaceutically acceptable salts of their components. Pharmaceutically acceptable salts include acid addition salts formed from inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, and mandelic acid (formed by the free amino group of the polypeptide). Salts formed by free carboxyl groups can also be obtained from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine. Physiologically acceptable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing an active ingredient (e.g., an antibody and / or its antigen-binding moiety or other binder) and water, which may contain a buffer such as sodium phosphate, physiological saline, or both, such as phosphate-buffered saline, at a physiological pH. Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts of sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also include a liquid phase in addition to and excluding water.Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0167] In some embodiments, a pharmaceutical composition comprising an antibody or its antigen-binding portion or other binder described herein, or a nucleic acid encoding an antibody or its antigen-binding portion or other binder, may be a lyophilized product.

[0168] In some embodiments, a syringe containing a therapeutically effective amount of the binder or pharmaceutical composition described herein is provided.

[0169] IV. Treatment methods and related uses In some embodiments, the binders described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) can be used in a manner comprising administering the binder or pharmaceutical composition described herein to a subject having an inflammatory disease.

[0170] In some embodiments, the binders described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) can be used in a manner that includes administering the binders or pharmaceutical compositions described herein to subjects having an autoimmune disease. In some embodiments, the autoimmune disease is a rheumatic disorder, a fibrous disorder, a gastrointestinal disorder, an endocrine disorder, a neurological disorder, or a skin disorder.In some embodiments, autoimmune diseases include autoimmune-induced hepatitis, Addison's disease, alopecia areata, Alport syndrome, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myositis, Behçet's disease, bird's-foot lung, bronchial asthma, Kaplan syndrome, cardiomyopathy, and celiac disease. Diseases) Chagas disease, chronic glomerulonephritis, chronic graft-versus-host disease, Cogan syndrome, cold agglutinin disease, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, lupus discoid, Dressiller syndrome, Eaton-Lambert syndrome, encephalomyelitis, endocrine ophthalmopathy, lupus erythematosus, Evans syndrome, Felty syndrome, fibromyalgia, Fuchs cyclitis, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Guts Dopasture syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré disease (syndrome), Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, hyperviscosity syndrome, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inflammatory bowel disease (syndrome), insulin-dependent diabetes mellitus (IDDM or type 1), juvenile arthritis, juvenile idiopathic arthritis, juvenile diabetes mellitus (type 1), Lambert-Eaton syndrome laminitis, lichen planus, lupoid hepatitis, Lupus, lupus nephritis, lymphopenia, macroglobulinemia, Meniere's disease, mixed connective tissue disease, monoclonal gammapathies of unknown cause, multiple sclerosis, amyotrophic lateral sclerosis (ALS), myasthenia gravis, myocarditis, pemphigus / bullous pemphigoid, pernicious anemia, POEMS syndrome, polyglandular syndrome, polyarteritis nodosa, polymyositis, presenile dementia, primary agammaglobulinemia, primary biliary cirrhosis / cholangitis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, These include rheumatic fever, rheumatoid arthritis, Sumpter syndrome, Schmidt syndrome, scleroderma / systemic sclerosis, Schulman syndrome, Sjögren's syndrome, Stiffman syndrome, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, type B insulin resistance, type I diabetes, ulcerative colitis, uveitis, vitiligo, Waldenström macroglobulinemia, and / or Wegener's granulomatosis.In some embodiments, the autoimmune disease is autoimmune hepatitis, celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes mellitus), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / bullous pemphigoid, primary biliary cirrhosis / cholangitis, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), or ulcerative colitis.

[0171] In some embodiments, the binders described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) can be used in a manner that includes administering the binders or pharmaceutical compositions described herein to a subject having celiac disease.

[0172] In some embodiments, the binders described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) can be used in a manner that includes administering the binders or pharmaceutical compositions described herein to a subject having type 1 diabetes.

[0173] In some embodiments, the binders described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) may be used in methods for treating graft-versus-host disease (GVHD)-related transplant complications, which include administering the binders or pharmaceutical compositions described herein to a target.

[0174] In some embodiments, the conjugates described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) can be used in a manner comprising administering the conjugate or pharmaceutical composition described herein to a subject to modulate an immune response to a target virus. In some embodiments, the conjugate or pharmaceutical composition is administered to suppress, reduce, or prevent an immune response to a virus. In some embodiments, the immune response to be suppressed, reduced, or prevented is an immune response to a virus or its antigenic moiety. In some embodiments, the virus is a viral vector, and the administration of the conjugate suppresses, reduces, or prevents the induction of an undesirable immune response associated with vector-mediated delivery of genetic material. The use of viral vectors, such as adeno-associated virus (AAV) vectors, for delivering the gene of interest is now an important tool in therapeutic approaches, including gene substitution, gene silencing, gene addition, and gene editing. However, host immune responses can limit the effectiveness of these approaches (Wang et al., Nat Rev Drug Discov 18, 358-378, 2019, https: / / doi.org / 10.1038 / s41573-019-0012-9). For example, the host may produce neutralizing antibodies against the vector capsid based on exposure to wild-type virus, blocking gene delivery. The host may also produce neutralizing antibodies against the vector capsid, limiting the effectiveness of re-administration of the vector in therapies requiring repeated doses. Furthermore, the host may initiate cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, clearing transduced cells. In a subset of "responsive patients," AAV-mediated gene delivery may be associated with inflammatory side effects and toxicity mediated by pathogenic CD4+ T cells.

[0175] Accordingly, the conjugates described herein (e.g., antibodies and their antigen-binding fragments, binding proteins) can be used in a manner comprising administering the conjugate or pharmaceutical composition described herein to a subject to suppress, reduce, or prevent an immune response to a viral vector. Where used herein, “immune response to a virus” or “immune response to a viral vector” may refer to any immune response to a virus, a viral vector, or its antigenic portion, such as a viral protein or fragment thereof. In some embodiments, the immune response may be the activation or proliferation of CD4+ T cells. The immune response may be characterized, for example, by the production of pro-inflammatory cytokines (e.g., IFN-γ) by CD4+ T cells. In some embodiments, the viral vector has been, is being, or is scheduled to be administered to the subject. In some embodiments, the immune response to the viral vector is induced by the administration of the viral vector to the subject. In some embodiments, the virus or viral vector is a retrovirus, adenovirus, parvovirus, coronavirus, orthomyxovirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, or hepatitis virus. In some embodiments, the virus or viral vector is an adeno-associated virus (AAV) vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, or AAVrh.37, or a variant thereof. In some embodiments, the virus or viral vector is an adenovirus vector. In some embodiments, the virus or viral vector is a lentiviral vector.

[0176] As used herein, “activating or stimulating” a CD8+ Treg or activated CD8+ Treg refers to an increase in the regulatory T cell function of such cells, e.g., their ability to suppress immune responses. Activation or stimulation of a CD8+ Treg may include the removal of the suppressive effect on such cells in order to restore the CD8+ Treg (e.g., restoring balance to the immune system or restoring balanced immune activity in a subject before receiving a viral vector). Activation or stimulation of a CD8+ Treg may also include the consequences of such activation or stimulation, including the removal of CD4+ cells, B cells, or other cells that mediate immune responses, e.g., cytolysis.

[0177] In some embodiments, CD8+ Tregs are contacted with a binder in vivo. In some embodiments, CD8+ Tregs are contacted with a binder ex vivo. The activated CD8+ Tregs can then be administered in an effective dose to the target subject requiring administration.

[0178] In some embodiments, activated CD8+ Tregs exert an inhibitory effect on CD4+ T cells, antibody-producing B cells, antigen-presenting dendritic cells, or other immune cells. In some embodiments, activated CD8+ Tregs exert an inhibitory effect on CD4+ T cells, antibody-producing B cells, and other immune cells. In some embodiments, activated CD8+ Tregs deplete other immune cells such as CD4 T cells, antibody-producing B cells, and antigen-presenting dendritic cells. In some embodiments, activated CD8+ Tregs modulate the activity of undesirable immune cells and reduce the titer of antibodies in the target. In some embodiments, activated CD8+ Tregs reduce the titer of antibodies in the target. In some embodiments, the CD8+ Tregs are CD39+ and KIR+.

[0179] In some embodiments, a pharmaceutical composition comprising a binder or any of the binders described herein is administered together with an immunosuppressant (e.g., a corticosteroid). In some embodiments, the immunosuppressant is one or more of the following: calcineurin inhibitors, e.g., cyclosporine or ascomycin, e.g., cyclosporine A (NEORAL®), FK506 (tacrolimus), pimecrolimus; mTOR inhibitors, e.g., rapamycin or its derivatives, e.g., sirolimus (RAPAMUNE®), everolimus (Certican®), temsirolimus, zotarolimus, biolimus-7, biolimus-9; rapalog, e.g., ridafololimus, azathioprine, campath 1H; S1P receptor modulators, e.g., fingolimod or its analogues; anti-IL-8 antibodies; mycophenolic acid or its salts, e.g., sodium salts, e.g., mycophenolate mofetil (CELLCEPT®), OKT3 (ORTHOCLONE) OKT3 (registered trademark), prednisone, ATGAM (registered trademark), THYMOGLOBULIN (registered trademark), Brekinal sodium, OKT4, T10B9.A-3A, 33B3.1,15-Deoxysperguarine, Tresperimus, Leflunomide ARAVA®, CTLAI-Ig, Anti-CD25, Anti-IL2R, Basiliximab (SIMULECT®), Daclizumab (ZENAPAX®), Misolvin, Methotrexate, Dexamethasone, ISAtx-247, SDZ ASM 981 (pimecrolimus, Elidel®), CTLA4Ig (abatacept), belatacept, LFA3Ig, etanercept (marketed as Enbrel® by Immunex), adalimumab (Humira®), infliximab (Remicade®), anti-LFA-1 antibody, natalizumab (Antegren®), enrimomab, gabirimomab, anti-thymocyte immunoglobulin, cyprizumab, alefacept, efalizumab, pentasa, mesala Zin, asacol, codeine phosphate, benolilate, fenbufen, naprosin, diclofenac, etodolac, indomethacin, tocilizumab (Actemra), siltuximab (Sylvant), secukibumab (Cosentyx), ustekinumab (Stelara), risankizumab, cifarimumab, aspirin, ibuprofen, immunofidase, proteasome inhibitors, arsenic trioxide, rabbit antithymocyte globulin (see, for example, Chu et al., Frontiers in Immunology 12:658038, 2021).

[0180] In some embodiments, a pharmaceutical composition of a binder (e.g., an antibody and its antigen-binding fragment, a binding protein) or any of the binders described herein is administered together with an anti-inflammatory agent, such as a corticosteroid. In some embodiments, the anti-inflammatory agent is one or more of the following: methotrexate, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, fluromethalone acetate, fluromethalone alcohol alcohol), rotoprendol etabonate, medrizone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone, rhodoxamide tromethamine, aspirin, ibuprofen, suprofen, piroxicam, meloxicam, flubiprofen, naproxan, ketoprofen, tenoxicam, diclofenac sodium, ketotifen fumarate, diclofenac sodium, nepafenac, bromfenac, flurbiprofen sodium, suprofen, celecoxib, naproxen, rofecoxib, glucocorticoids, diclofenac, and any combination thereof. In some embodiments, the anti-inflammatory agent is one or more nonsteroidal anti-inflammatory drugs (NSAIDs), such as naproxen sodium (Anaprox), celecoxib (Celebrex), sulindac (Clinoril), oxaprozin (Daypro), sarsalate (Disalcid), diflunisal (Dolobid), piroxicam (Feldene), indomethacin (Indocin), etodolac (Lodine), meloxicam (Mobic), naproxen (Naprosyn), nabumetone (Relafen), ketrolactromethamine (Toradol), naproxen / esomeprazole (Vimovo), and diclofenac (Voltaren), as well as combinations thereof.

[0181] In some embodiments of the methods described above, the binder (e.g., an antibody or its antigen-binding fragment, binding protein) is administered in doses of about 0.01 mg / kg to about 20 mg / kg (i.e., about 0.01 mg to about 20 mg of binder per kg of body weight). In some embodiments, the binder is administered in doses of about 0.5 mg / kg to about 15 mg / kg. In some embodiments, the binder is administered in doses of about 0.5 mg / kg to about 5 mg / kg. In some embodiments, the binder is administered in doses of about 0.01 mg / kg to about 10 mg / kg. In some embodiments, the binder is administered in doses of about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the binder is administered in doses of about 0.5 mg / kg to about 10 mg / kg. In some embodiments, the binder is administered in doses of about 0.05 mg / kg to about 0.1 mg / kg. In some embodiments, the binder is administered in doses of approximately 0.5 mg / kg to approximately 1.0 mg / kg. In some embodiments, the binder is administered in doses up to approximately 10 mg / kg. In some embodiments, the binder is administered in doses of approximately 10 mg / kg. In some embodiments, the binder is administered in doses of 0.01 mg / kg to 20 mg / kg. In some embodiments, the binder is administered in doses of 0.5 mg / kg to 15 mg / kg. In some embodiments, the binder is administered in doses of 0.5 mg / kg to 5 mg / kg. In some embodiments, the binder is administered in doses of 0.01 mg / kg to 10 mg / kg. In some embodiments, the binder is administered in doses of 0.1 mg / kg to 10 mg / kg. In some embodiments, the binder is administered in doses of 0.5 mg / kg to 10 mg / kg. In some embodiments, the binder is administered in doses of 0.05 mg / kg to 0.1 mg / kg. In some embodiments, the binder is administered at a dose of 0.5 mg / kg to 1.0 mg / kg. In some embodiments, the binder is administered at a maximum dose of 10 mg / kg. In some embodiments, the binder is administered at a dose of 10 mg / kg. In some embodiments, the binder is administered at a dose of approximately 50 mg / kg. In some embodiments, the binder is administered at a dose of 50 mg / kg.In some embodiments, the binder is administered at a dose of 0.01 mg / kg. In some embodiments, the binder is administered at a maximum dose of 0.1 mg / kg. In some embodiments, the binder is administered at a dose of 0.1 mg / kg. In some embodiments, the binder is administered at a maximum dose of 0.5 mg / kg. In some embodiments, the binder is administered at a dose of 0.5 mg / kg. In some embodiments, the binder is administered at a maximum dose of 2.0 mg / kg. In some embodiments, the binder is administered at a dose of 2.0 mg / kg. In some embodiments, the binder is administered at a maximum dose of 8.0 mg / kg. In some embodiments, the binder is administered at a dose of 8.0 mg / kg. In some embodiments, the binder is administered in multiple doses, for example, two doses. In some embodiments, the dose range can be set to maintain serum levels between 0.1 ug / mL and 1000 ug / mL.

[0182] In some embodiments, the binder is administered by intravenous infusion.

[0183] In some embodiments, the binders disclosed herein (e.g., antibodies and their antigen-binding fragments, binding proteins) are for use in the aforementioned methods or for use in the manufacture of pharmaceuticals for use in the aforementioned methods.

[0184] V. Exemplary Embodiments Exemplary embodiments include, but are not limited to, the following:

[0185] Embodiment 1. [ka] It includes a light chain variable region (VL) that has at least 90% identity with the amino acid sequence of the , Here, X is a binding protein where X = G or C.

[0186] Embodiment 2. The binding protein according to Embodiment 1, wherein the VL has the CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0187] Embodiment 3. The binding protein according to Embodiment 1, wherein the VL has the CDRL1, CDRL2, and CDRL3 amino acid sequences according to one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

[0188] Embodiment 4. [ka] A binding protein comprising a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of the specified protein, where (a) X1=I or F, (b) X2=G or C, (c) X3=F or V, (d) X4=A or F, (e) X5=A or R, and (f) X6=G or A.

[0189] Embodiment 5. The binding protein according to Embodiment 4, wherein the VH has the CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0190] Embodiment 6. The binding protein according to Embodiment 4, wherein the VH has the CDRH1, CDRH2, and CDRH3 amino acid sequences according to one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

[0191] It is a binding protein,

[0192] (a) [ka] comprising at least 90% identity with the amino acid sequence according to, wherein X = G or C, a light chain variable region (VL), and

[0193] (b)

Chemical formula

[0194] Embodiment 8. The binding protein according to embodiment 7, wherein the VL has the CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0195] Embodiment 9. The binding protein according to embodiment 7, wherein the VL has the CDRL1, CDRL2, and CDRL3 amino acid sequences according to any one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

[0196] Embodiment 10. The binding protein according to any one of embodiments 7 to 9, wherein the VH has the CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0197] Embodiment 11. The binding protein according to any one of embodiments 7 to 9, wherein the VH has the CDRH1, CDRH2, and CDRH3 amino acid sequences according to any one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

[0198] Embodiment 12. A binding protein comprising (a) the amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and (b) the following framework regions: (i) light chain FR4 according to SEQ ID NO: 126, (ii) heavy chain FR2 according to SEQ ID NO: 128, (iii) heavy chain FR3 according to SEQ ID NO: 129, (iv) heavy chain FR3 according to SEQ ID NO: 130, (v) heavy chain FR3 according to SEQ ID NO: 131, (vi) heavy chain FR3 according to SEQ ID NO: 132, and (vii) one or more of the following substitutions at the position of an amino acid in SEQ ID NO: 16, one or more of the heavy chain FR3 according to SEQ ID NO: 16, one or more of the following: F6V, A10F, A14R, and G39A.

[0199] Embodiment 13. A binding protein comprising (a) the amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO: 127, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and (b) the following framework regions: (i) light chain FR4 according to SEQ ID NO: 126, (ii) heavy chain FR2 according to SEQ ID NO: 128, (iii) heavy chain FR3 according to SEQ ID NO: 129, (iv) heavy chain FR3 according to SEQ ID NO: 130, (v) heavy chain FR3 according to SEQ ID NO: 131, (vi) heavy chain FR3 according to SEQ ID NO: 132, (vii) heavy chain FR3 according to SEQ ID NO: 16 having one or more of the following substitutions at the position of an amino acid in SEQ ID NO: 16: F6V, A10F, A14R, and G39A, (viii) the light chain framework region of VL according to SEQ ID NO: 7, and (ix) the heavy chain framework region of VH according to SEQ ID NO: 8.

[0200] Embodiment 14. A binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 55 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 56, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 55 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 56, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 55 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 56.

[0201] Embodiment 15. A binding protein comprising: (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 18; (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 18; or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 18.

[0202] Embodiment 16. A binding protein comprising: (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 20; (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 20; or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 20.

[0203] Embodiment 17. A binding protein comprising: (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 22; (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 22; or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 22.

[0204] Embodiment 18. A binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 24, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 24, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 24.

[0205] Embodiment 19. A binding protein,

[0206] A binding protein comprising: (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 26; (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 26; or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 26.

[0207] Embodiment 20. A binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 27 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 28, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 27 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 28, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 27 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 28.

[0208] Embodiment 21. A binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 30, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 30, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 30.

[0209] Embodiment 22. A binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 32, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 32, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 32.

[0210] Embodiment 23. A binding protein comprising (a) a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 33 and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 34, (b) a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 33 and a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 34, or (c) a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 33 and a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 34.

[0211] Embodiment 24. A binding protein comprising (a) a light chai...

Claims

1. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 55, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 56, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 55, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 56, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 55, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

56. 【Request Item 2】 【Chemistry 7】 It includes a light chain variable region (VL) that has at least 90% identity with the amino acid sequence of the following: Here, X = G or C, which is the binding protein.

3. The binding protein according to claim 2, wherein the VL has the CDRL1, CDRL2, and CDRL3 amino acid sequences of RTSRSISQYLA (SEQ ID NO: 1), SGSTLQS (SEQ ID NO: 2), and QQHNENPLT (SEQ ID NO: 3).

4. The binding protein according to claim 2, wherein the VL has CDRL1, CDRL2, and CDRL3 amino acid sequences derived from one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo. 【Request Item 5】 【Chemistry 8】 It includes a heavy chain variable region (VH) that has at least 90% identity with the amino acid sequence, where, (a) X １ = I or F, (b) X ２ = G or C, (c) X ３ = F or V, (d) X ４ = A or F, (e) X ５ = A or R, and (f) X ６ = A binding protein that is either G or A.

6. The binding protein according to claim 5, wherein the VH has the CDRH1, CDRH2, and CDRH3 amino acid sequences of GFNIKDT (SEQ ID NO: 4), RIDPANDNT (SEQ ID NO: 5), and GYYVFDH (SEQ ID NO: 6), respectively.

7. The binding protein according to claim 5, wherein the VH has CDRH1, CDRH2, and CDRH3 amino acid sequences derived from one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

8. It is a binding protein, (a) 【Chemistry 9】 It contains at least 90% identity with the amino acid sequence, Here, the light chain variable region (VL) is such that X = G or C, (b) 【Chemistry 10】 It contains at least 90% identity with the amino acid sequence, Here, (1) X １ = I or F, (2) X ２ = G or C, (3) X ３ = F or V, (4) X ４ = A or F, (5) X ５ = A or R, and (6) X ６ A binding protein containing a heavy chain variable region (VH) which is either G or A.

9. The binding protein according to claim 8, wherein the VL has the CDRL1, CDRL2, and CDRL3 amino acid sequences according to RTSRSISQYLA (SEQ ID NO: 1), SGSTLQS (SEQ ID NO: 2), and (QQHNENPLT) SEQ ID NO:

3.

10. The binding protein according to claim 8, wherein the VL has CDRL1, CDRL2, and CDRL3 amino acid sequences derived from one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

11. The binding protein according to any one of claims 8 to 10, wherein the VH has the CDRH1, CDRH2, and CDRH3 amino acid sequences of GFNIKDT (SEQ ID NO: 4), RIDPANDNT (SEQ ID NO: 5), and GYYVFDH (SEQ ID NO: 6), respectively.

12. The binding protein according to any one of claims 8 to 10, wherein the VH has CDRH1, CDRH2, and CDRH3 amino acid sequences derived from one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

13. It is a binding protein, (a) The amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, (b) The following framework areas: (i) Light chain FR4 according to Sequence ID No. 126, (ii) Heavy chain FR2 according to Sequence ID No. 128, (iii) Heavy chain FR3 according to Sequence ID No. 129, (iv) Heavy chain FR3 according to Sequence ID No. 130, (v) Heavy chain FR3 according to Sequence ID No. 131, (vi) Heavy chain FR3 according to Sequence ID No. 132, and (vii) The following substitutions based on the position of amino acids in SEQ ID NO: 16: Heavy chain FR3 by SEQ ID NO: 16 having one or more of F6V, A10F, A14R, and G39A A binding protein containing one or more of the following.

14. It is a binding protein, (a) The amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO: 127, SEQ ID NO: 5, and SEQ ID NO: 6, (b) The following framework areas: (i) Light chain FR4 according to Sequence ID No. 126, (ii) Heavy chain FR2 according to Sequence ID No. 128, (iii) Heavy chain FR3 according to Sequence ID No. 129, (iv) Heavy chain FR3 according to Sequence ID No. 130, (v) Heavy chain FR3 according to Sequence ID No. 131, (vi) Heavy chain FR3 according to Sequence ID No. 132, (vii) The following substitutions based on the position of amino acids in SEQ ID NO: 16: Heavy chain FR3 by SEQ ID NO: 16 having one or more of F6V, A10F, A14R, and G39A. (viiii) The light chain framework region of VL according to Sequence ID No. 7, and (ix) Heavy chain framework region of VH according to Sequence ID 8 A binding protein containing one or more of the following.

15. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 17, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 18, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 17, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 18, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 17, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

18.

16. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 20, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 19, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 20, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 19, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

20.

17. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence according to SEQ ID NO: 21, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence according to SEQ ID NO:

22. (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 21, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 22, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 21, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

22.

18. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence according to SEQ ID NO: 23, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence according to SEQ ID NO:

24. (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 23, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 24, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 23, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

24.

19. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 26, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 25, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 26, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 25, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

26.

20. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 27, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 28, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 27, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 28, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 27, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

28.

21. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 29, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 30, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 29, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 30, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 29, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

30.

22. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence according to SEQ ID NO: 31, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence according to SEQ ID NO:

32. (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 31, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 32, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 31, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

32.

23. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 33, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 34, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 33, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 34, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 33, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

34.

24. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 35, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 36, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 35, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 36, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 35, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

36.

25. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 37, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO:

38. (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 37, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 38, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 37, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

38.

26. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 39, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 40, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 39, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 40, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 39, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

40.

27. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 41, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 42, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 41, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 42, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 41, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

42.

28. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 43, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 44, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 43, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 44, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 43, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

44.

29. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 45, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 46, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 45, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 46, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 45, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

46.

30. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 47, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 48, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 47, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 48, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 47, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

48.

31. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 49, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 50, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 49, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 50, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 49, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

50.

32. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 51, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO:

52. (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 51, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 52, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 51, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

52.

33. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 53, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 54, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 53, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 54, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 53, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

54.

34. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 57, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO:

58. (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 57, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 58, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 57, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

58.

35. It is a binding protein, (a) A light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 59, and a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 60, (b) A light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 59, and a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 60, or (c) A binding protein comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 59, and a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

60.

36. The binding protein according to any one of claims 1 to 35, wherein the binding protein is scFv.

37. The binding protein according to any one of claims 1 to 36, wherein the VH and VL are linked by a linker polypeptide.

38. The binding protein according to claim 37, wherein the linker polypeptide comprises or comprises the amino acid sequence according to SEQ ID NO:

61.

39. The binding protein according to any one of claims 36 to 38, further comprising a constant domain containing the sequence described in Sequence ID No.

62.

40. The binding protein according to any one of claims 1 to 39, further comprising a polypeptide that binds to the KIR protein.

41. The binding protein according to any one of claims 1 to 39, further comprising a second polypeptide containing the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, RASQSVSSYLA (SEQ ID NO: 63), DASNRAT (SEQ ID NO: 64), and QQRSNWMYTF (SEQ ID NO: 65).

42. The binding protein according to claim 41, further comprising a third polypeptide containing the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, as defined by FYAIS (SEQ ID NO: 66), GFIPIFGAANYAQKF (SEQ ID NO: 67), and IPSGSYYYDYDMDV (SEQ ID NO: 68).

43. (a) A second polypeptide comprising a light chain variable region (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO: 69, and a third polypeptide comprising a heavy chain variable region (VH) having at least 90% identity with the amino acid sequence of SEQ ID NO: 70; (b) A second polypeptide comprising a light chain variable region (VL) containing the amino acid sequence according to SEQ ID NO: 69, and a third polypeptide comprising a heavy chain variable region (VH) containing the amino acid sequence according to SEQ ID NO: 70, or (c) The binding protein according to any one of claims 1 to 39, further comprising a second polypeptide comprising a light chain variable region (VL) consisting of the amino acid sequence according to SEQ ID NO: 69, and a third polypeptide comprising a heavy chain variable region (VH) consisting of the amino acid sequence according to SEQ ID NO:

70.

44. The binding protein according to claim 43, wherein the VL of the second polypeptide has the CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs. 63, 64, and 65, respectively, and the VH of the third polypeptide has the CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NOs. 66, 67, and 68, respectively.

45. The binding protein according to claim 43, wherein the VL of the second polypeptide has CDRL1, CDRL2, and CDRL3 amino acid sequences from one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

46. The binding protein according to claim 43 or claim 45, wherein the VH of the third polypeptide has CDRH1, CDRH2, and CDRH3 amino acid sequences from one of Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo.

47. The binding proteins listed in Table 3.

48. It is a binding protein, (a) a polypeptide sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 78, a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 80, and a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 120; (b) a polypeptide sequence containing the amino acid sequence according to SEQ ID NO: 78, a polypeptide containing the amino acid sequence according to SEQ ID NO: 80, and a polypeptide containing the amino acid sequence according to SEQ ID NO: 120; or (c) A binding protein comprising a polypeptide sequence consisting of the amino acid sequence according to SEQ ID NO: 78, a polypeptide consisting of the amino acid sequence according to SEQ ID NO: 80, and a polypeptide consisting of the amino acid sequence according to SEQ ID NO:

120.

49. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 48 and a pharmaceutically acceptable carrier.

50. A nucleic acid encoding the binding protein according to any one of claims 1 to 48.

51. Nucleic acid molecules, including sequence numbers 139, 141, and 141.

52. Nucleic acid molecules, including sequence numbers 138, 140, and 142.

53. A vector comprising the nucleic acid described in any one of claims 50 to 52.

54. A cell line comprising the vector described in claim 53.

55. A method for treating a disease, comprising administering a binding protein according to any one of claims 1 to 48 or a pharmaceutical composition according to claim 49 to a subject in need thereof.

56. A method for preventing a disease, comprising administering a binding protein according to any one of claims 1 to 48 or a pharmaceutical composition according to claim 49 to a subject in need thereof.

57. The method according to claim 55 or claim 56, wherein the disease is an inflammatory disease or an autoimmune disease.

58. The method according to claim 55 or claim 66, wherein the disease is a CD4+ T cell-driven inflammatory disease or an autoimmune disease.

59. The method according to claim 57 or claim 58, wherein the number or activity of pathogenic immune cells in the subject is reduced.

60. The method according to any one of claims 55 to 59, wherein the disease is a rheumatic disorder, a fibrous disorder, a gastrointestinal disorder, an endocrine disorder, a neurological disorder, or a skin disorder.

61. The method according to any one of claims 57 to 59, wherein the autoimmune disease is celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes mellitus), lupus, lupus nephritis, cutaneous lupus, discoid lupus, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / bullous pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), or ulcerative colitis.

62. The method according to any one of claims 57 to 59, wherein the autoimmune disease is celiac disease.

63. The method according to any one of claims 57 to 59, wherein the autoimmune disease is Crohn's disease.

64. The method according to any one of claims 57 to 59, wherein the autoimmune disease is inflammatory bowel disease (IBD).

65. The method according to any one of claims 57 to 59, wherein the autoimmune disease is ulcerative colitis.

66. The method according to any one of claims 55 to 59, wherein the disease is graft-versus-host disease (GVHD).

67. The method according to any one of claims 55 to 59, wherein the disease is type 1 diabetes.

68. A method for suppressing an immune response mediated by pathogenic immune cells, comprising contacting CD8+ regulatory T cells (Treg) with a binding protein according to any one of claims 1 to 48 or a pharmaceutical composition according to claim 49, thereby activating or stimulating the CD8+ Treg.

69. A method for suppressing an immune response to an antigen, such as an autoantigen, comprising administering to a subject in need thereof a binding protein according to any one of claims 1 to 48 or a pharmaceutical composition according to claim 49, thereby activating or stimulating CD8+ Treg, thereby reducing the number or activity of pathogenic immune cells that respond to the antigen or autoantigen.

70. The method according to claim 68 or claim 69, wherein the pathogenic immune cells are autoreactive CD4+ T cells, autoantibody-producing B cells, or autoantigen-presenting dendritic cells.

71. The method according to claim 68 or claim 69, wherein the pathogenic immune cells are autoreactive CD4+ T cells.

72. The method according to any one of claims 68 to 71, wherein the CD8+Treg is CD8+KIR+Treg.

73. The method according to any one of claims 68 to 72, wherein the activated CD8+ Treg is administered to the subject or a second subject.

74. The method according to any one of claims 68 to 73, wherein the subject and / or the second subject has an inflammatory disease or an autoimmune disease.

75. A method for suppressing, reducing, or preventing an immune response to a viral vector in a subject, comprising administering to the subject a binding protein according to any one of claims 1 to 48 or a pharmaceutical composition according to claim 49.

76. The method according to claim 75, wherein the viral vector has been administered to, will be administered to, or will be administered to the subject, and the immune response to the viral vector is induced by administering the viral vector to the subject.

77. The method according to any one of claims 55 to 76, wherein the binding protein is administered in a dose of about 0.01 mg / kg to about 20 mg / kg.

78. The method according to claim 77, wherein the binding protein is administered in a dose of about 0.01 mg / kg to about 10 mg / kg.

79. The method according to claim 77, wherein the binding protein is administered in a dose of about 0.05 mg / kg to about 0.1 mg / kg.

80. The method according to claim 77, wherein the binding protein is administered in a dose of about 0.5 mg / kg to about 1.0 mg / kg.

81. The method according to claim 77, wherein the binding protein is administered in a dose of 0.05 mg / kg to 0.1 mg / kg.

82. The method according to claim 77, wherein the binding protein is administered in a dose of 0.5 mg / kg to 1.0 mg / kg.

83. The method according to claim 77, wherein the binding protein is administered at a maximum dose of approximately 10 mg / kg.

84. The method according to claim 77, wherein the binding protein is administered at a dose of up to 10 mg / kg.

85. The method according to claim 77, wherein the binding protein is administered at a dose of approximately 10 mg / kg.

86. The method according to claim 77, wherein the binding protein is administered at a dose of 10 mg / kg.

87. The method according to any one of claims 55 to 86, using the binding protein according to any one of claims 1 to 48 or the pharmaceutical composition according to claim 49.

88. A binding protein according to any one of claims 1 to 48 or a pharmaceutical composition according to claim 49, for use in the method according to any one of claims 55 to 86.

89. Use of the binding protein according to any one of claims 1 to 48 or the pharmaceutical composition according to claim 49 in the manufacture of a pharmaceutical for use in the method according to any one of claims 55 to 86.

90. A method for treating celiac disease in a subject requiring treatment for celiac disease, comprising administering the binding protein described in claim 48 to the subject.

91. A method for preventing celiac disease in a subject requiring prevention of celiac disease, comprising administering the binding protein described in claim 48 to the subject.

92. A method for treating celiac disease in a subject requiring treatment for celiac disease, comprising administering the pharmaceutical composition according to claim 49 to the subject.

93. A method for preventing celiac disease in a subject requiring prevention of celiac disease, comprising administering the pharmaceutical composition described in claim 49 to the subject.

94. A method for treating type 1 diabetes in a subject requiring treatment for type 1 diabetes, comprising administering the binding protein described in claim 48 to the subject.

95. A method for preventing type 1 diabetes in a subject requiring prevention of type 1 diabetes, comprising administering the binding protein described in claim 48 to the subject.

96. A method for treating type 1 diabetes in a subject requiring treatment for type 1 diabetes, comprising administering the pharmaceutical composition described in claim 49 to the subject.

97. A method for preventing type 1 diabetes in a subject requiring prevention of type 1 diabetes, comprising administering the pharmaceutical composition described in claim 49 to the subject.

98. The use of the binding protein according to claim 48 or the pharmaceutical composition according to claim 49 in the method according to any one of claims 90 to 97.

99. A binding protein according to claim 48 or a pharmaceutical composition according to claim 49 for use in the method according to any one of claims 90 to 97.

100. Use of the binding protein according to claim 48 or the pharmaceutical composition according to claim 49 in the manufacture of a pharmaceutical for use in the method according to any one of claims 90 to 97.

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