Anti-CD19 antibody-drug conjugates

Abstract

To provide an agent that delivers a glucocorticoid modulator to treat diffuse large B-cell lymphoma (DLBCL), acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL).SOLUTION: The present invention provides antibody-drug conjugates that target B-cells via an anti-CD19 antibody linked to a glucocorticoid receptor modulator (GRM) agonist payload.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 366,520, filed June 16, 2022, and U.S. Provisional Patent Application No. 63 / 488,302, filed March 3, 2023, the entire disclosures of which are incorporated herein by reference for all purposes.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .xml format and is incorporated herein by reference in its entirety. The .xml copy was created on May 10, 2024, is named SeqList-350794-46710, and is 21,125 bytes in size.

[0003] 3.Technical Field This application relates, inter alia, to novel anti-CD19 antibody drug conjugates (ADCs) and methods for making same. [Background technology]

[0004] 4.Background CD19 is a B cell marker and a coactivator of the B cell receptor (BCR) complex, which activates downstream proteins such as PI3K, thereby resulting in a proliferative phenotype. Due to this mechanism, CD19 is an essential B cell antigen, and loss of CD19 expression by CRISPR or RNAi renders normal B cells and B cell cancer lines nonviable. CD19 is a clinically validated target with high expression across B cell malignancies (e.g., DLBCL, FL, CLL).

[0005] Systemic glucocorticoids are used to treat B-cell malignancies. While these systemic steroids have robust monotherapy activity at high doses, chronic steroid-related toxicity can occur, limiting their potential for dosing and efficacy. Glucocorticoids function by binding to the intracellular glucocorticoid receptor (GR), which then transports it to the nucleus, where it binds to the glucocorticoid response element (GRE) and drives transcriptional activation. Steroid-induced transcriptional changes include upregulation of proapoptotic proteins (e.g., BIM, BAX), reduction of antiapoptotic proteins (e.g., BCL2), and inhibition of NFκB proliferation signaling, ultimately leading to apoptotic cancer cell death. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need in the art for agents that deliver glucocorticoid modulators to treat diffuse large B-cell lymphoma (DLBCL), acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). [Means for solving the problem]

[0007] (Summary of the Invention) 5. Summary The present invention provides antibody-drug conjugates that target B cells via anti-CD19 antibodies linked to glucocorticoid receptor modulator (GRM) agonist payloads and offers a novel approach for treating diffuse large B-cell lymphoma (DLBCL), acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). The anti-CD19 GRM agonist ADCs described herein are engineered to possess distinct mechanisms of action: (1) targeted delivery of a highly potent glucocorticoid receptor modulator (GRM) agonist payload, (2) inhibition of CD19 signaling, and (3) Fc effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), via CD19 antibody defucosylation. Unlike conventional cytotoxic ADCs, the GRM payload is an agonist that binds to the intracellular glucocorticoid receptor (GR), which then transports it to the nucleus and drives sustained glucocorticoid response element (GRE)-associated transcriptional events, including modulation of apoptotic regulators, resulting in payload-driven apoptotic cell death. ADCC is a highly effective mechanism in B-cell malignancies. In some embodiments, the anti-CD19 antibody of the ADC is engineered to maintain enhanced ADCC activity after conjugation with the GRM agonist payload. By engineering the molecule in this way, the anti-CD19 GRM agonist ADC becomes a versatile antibody-drug conjugate designed to have robust single-agent activity and provide significant clinical benefit against a variety of B-cell malignancies.

[0008] In certain aspects, the present invention provides a compound having the following structure:

[0009] [ka] (In the formula, A is an antibody, R is the point of attachment of the antibody via a cysteine ​​residue on the antibody, which upon linkage provides an -S- group; AA1 and AA2 are alanine (Ala), R 1 is hydrogen or -P(=O)(OH)2, n is an integer from 1 to 10. 1. An anti-CD19 antibody drug conjugate comprising: The antibody, a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and a light chain variable region comprising the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain an anti-CD19 antibody comprising CDR-H1 comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1), CDR-H2 comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2), CDR-H3 comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3), CDR-L1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4), CDR-L2 comprises the amino acid sequence WASTRHT (SEQ ID NO: 5), and An anti-CD19 antibody drug conjugate is provided, wherein CDR-L3 comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6).

[0010] 6. Brief description of the drawings The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows an in vitro cytotoxicity assay of GRM payloads compared to approved small molecule steroids (dexamethasone and prednisolone). The % cell viability of RS4;11 after 5 days of compound treatment compared to vehicle control is shown. The EC50 for each compound is summarized in the table. [Figure 2] Photographs showing that CD19 mAb induces internalization of CD19 into lysosomes. Immunofluorescence staining showing localization of CD19 receptor to the lysosomal compartment in RS4;11 after treatment with CD19 mAb. LAMP-1 is used as a marker for the lysosomal compartment. [Figure 3]

[0023] Figure 1 shows that ADC-1 induces transcriptional activation of a glucocorticoid response element (GRE) luciferase reporter. K562 cells expressing human CD19 were treated with ADC-1 for 72 hours. % maximal GRE activation was calculated by dividing the GRE luminescence signal at each concentration by the GRE luminescence signal at 333 nM. [Figure 4A] Figure 1 shows that CD19 mAb inhibits DLBCL cell proliferation and AKT activation in vitro.% viability of SU-DHL-6 compared to vehicle control after 5 days of treatment with isotype or CD19 mAb. [Figure 4B] (Figure 1 shows that CD19 mAb inhibits DLBCL cell proliferation and AKT activation in vitro. SU-DHL-6 cells were treated with isotype or CD19 mAb for 1 hour and then stimulated with 1 μg / ml anti-IgM for the indicated times before cytolysis. Phosphorylation of S473 of AKT was detected by immunoblotting, and GAPDH was used as a loading control. [Figure 5A]Figures 1A and 1B show that CD19 mAb and ADC-1 induce NFAT activation in Jurkat reporter cells expressing the FcγRIIIa V158 and F158 allotypes. A and B, Raji cells were treated with titrated antibodies or ADCs and co-incubated with Jurkat cells expressing the V158 (A) and F158 (B) FcγRIIIa variants. Luminescence signals are shown after 4 hours (A) or 16 hours (B) of incubation. [Figure 5B] Figures 1A and 1B show that CD19 mAb and ADC-1 induce NFAT activation in Jurkat reporter cells expressing the FcγRIIIa V158 and F158 allotypes. A and B, Raji cells were treated with titrated antibodies or ADCs and co-incubated with Jurkat cells expressing the V158 (A) and F158 (B) FcγRIIIa variants. Luminescence signals are shown after 4 hours (A) or 16 hours (B) of incubation. [Figure 6A] Figures 1A-C show that CD19 mAb and ADC-1 induce ADCC against B-cell malignant cell lines in an ex vivo PBMC co-culture ADCC assay. RS4;11 (A), Raji (B), and KARPAS422 (C) were incubated with titrated antibodies or ADCs and co-incubated with primary PBMCs at an effector-target ratio (E:T) of 20:1 for 4 hours. % specific lysis of cells was calculated relative to untreated samples. [Figure 6B] Figures 1A-C show that CD19 mAb and ADC-1 induce ADCC against B-cell malignant cell lines in an ex vivo PBMC co-culture ADCC assay. RS4;11 (A), Raji (B), and KARPAS422 (C) were incubated with titrated antibodies or ADCs and co-incubated with primary PBMCs at an effector-target ratio (E:T) of 20:1 for 4 hours. % specific lysis of cells was calculated relative to untreated samples. [Figure 6C]Figures 1A-C show that CD19 mAb and ADC-1 induce ADCC against B-cell malignant cell lines in an ex vivo PBMC co-culture ADCC assay. RS4;11 (A), Raji (B), and KARPAS422 (C) were incubated with titrated antibodies or ADCs and co-incubated with primary PBMCs at an effector-target ratio (E:T) of 20:1 for 4 hours. % specific lysis of cells was calculated relative to untreated samples. [Figure 7A] Figures 1A and 1B show that ADC-1 induces antibody-dependent cellular phagocytosis (ADCP) of B-cell malignant cell lines in coculture with monocyte-derived macrophages. (A and B) CFSE-labeled NuDHL1 cells (A) and Raji cells (B) were treated with 200 nM of the indicated agents and cocultured with monocyte-derived macrophages for 3 hours. CD68+ was used as a marker for mature macrophages, and the phagocytosis index represents the percentage of phagocytosed target cells (CFSE+CD68+) among the total mature macrophage (CD68+) gate. [Figure 7B] Figures 1A and 1B show that ADC-1 induces antibody-dependent cellular phagocytosis (ADCP) of B-cell malignant cell lines in coculture with monocyte-derived macrophages. (A and B) CFSE-labeled NuDHL1 cells (A) and Raji cells (B) were treated with 200 nM of the indicated agents and cocultured with monocyte-derived macrophages for 3 hours. CD68+ was used as a marker for mature macrophages, and the phagocytosis index represents the percentage of phagocytosed target cells (CFSE+CD68+) among the total mature macrophage (CD68+) gate. [Figure 8AB]Graphs showing growth inhibition of xenografted diffuse large B-cell lymphoma (OCI-LY19 and SU-DHL-6) in immunocompromised mice by various doses of ADC-1. A and B, Growth inhibition of xenografted OCI-LY19 tumors grown in CB17 / SCID mice (A) and SU-DHL-6 (B) cells by intraperitoneal (IP) administration of ADC-1. After randomization and dosing, subcutaneous tumor volume was plotted as a function of time. Growth inhibition of OCI-LY19 tumors (A) and SU-DHL-6 tumors (B) grown in SCID-beige mice was tested in mice bearing subcutaneous tumors (mean tumor volume 117-205 mm3) by ADC-1, isotype-GRM, huCD19 antibody, and isotype antibody administered as a single dose (QD x 1) at various dose levels (doses 1-5; dose 2 is 3x the dose of dose 1, dose 3 is 10x the dose of dose 1, dose 4 is 30x the dose of dose 1, and dose 5 is 50x the dose of dose 1). GRM-SM was administered as multiple daily doses (QD x 5). Each point on the curve represents the mean value of seven (SU-DHL-6) or eight (OCI-LY19) tumors. Error bars indicate the standard error of the mean. [Figure 9] 1 is a graph showing the growth inhibition of xenografted acute lymphoblastic leukemia (RS4;11) by various doses of ADC-1. Growth inhibition of xenografted RS4;11 tumors (immunocompromised CB17 / SCID mice) by administration of ADC-1. After randomization and dosing, subcutaneous tumor volume was plotted as a function of time. Growth inhibition of xenografted RS4;11 cells in mice bearing subcutaneous tumors (mean tumor volume 118 mm3) was tested by administration of ADC-1, isotype-GRM, as a single dose (QD x 1) at various doses (Dose 1 and Dose 2, where Dose 2 is twice the dose of Dose 1). Each point on the curve represents the average of five tumors. Error bars indicate the standard error of the mean. [Figure 10A]Figure 1 shows graphs showing growth inhibition of xenografted diffuse large B-cell lymphoma cells (OCI-LY19) by various doses of ADC-1 in humanized immunocompetent huCD34+ NSG huIL-15 mice. A) Growth inhibition of xenografted OCI-LY19 tumors by IP administration of isotype mAb, CD19 mAb, and ADC-1. ADC-1 was administered as a single dose (QD x 1) at various dose levels (doses 1-3, with dose 2 being 3x the dose of dose 1 and dose 3 being 10x the dose of dose 1). Each point on the curve represents the mean value of four or five tumors. Error bars indicate the standard error of the mean. B) Flow cytometry data for human B cells and human NK cells from tail vein bleeds obtained at the indicated time points. Each point on the curve represents the mean value of four or five animals. Error bars indicate the standard deviation of the mean. [Figure 10B] Figure 1 shows graphs showing growth inhibition of xenografted diffuse large B-cell lymphoma cells (OCI-LY19) by various doses of ADC-1 in humanized immunocompetent huCD34+ NSG huIL-15 mice. A) Growth inhibition of xenografted OCI-LY19 tumors by IP administration of isotype mAb, CD19 mAb, and ADC-1. ADC-1 was administered as a single dose (QD x 1) at various dose levels (doses 1-3, with dose 2 being 3x the dose of dose 1 and dose 3 being 10x the dose of dose 1). Each point on the curve represents the mean value of four or five tumors. Error bars indicate the standard error of the mean. B) Flow cytometry data for human B cells and human NK cells from tail vein bleeds obtained at the indicated time points. Each point on the curve represents the mean value of four or five animals. Error bars indicate the standard deviation of the mean. [Figure 11]This graph shows that ADC-1 induces superior antitumor activity compared with high-dose CD19 mAb, prednisolone alone, and the combination of CD19 mAb and prednisolone. Growth inhibition of xenografted RS4;11 tumors in CB17 / SCID mice by administration of the indicated treatments. After randomization and dosing, subcutaneous tumor volume was plotted as a function of time. Fucosylated mAb and ADC-1 were used in this study. Various dose levels were used (doses 1-3, with dose 2 being 10 times the dose of dose 1 and dose 3 being 33 + 1 / 3 times the dose of dose 1). Each point on the curve represents the average of five tumors. Error bars indicate the standard error of the mean. [Figure 12A] Figure 1 shows graphs demonstrating in vivo antitumor activity in multiple xenograft models of B-cell malignancies compared with prednisolone or isotype GRM controls. A-C, DB (A), SUPB15 (B), and RS4;11 (C) xenograft models were treated with the indicated agents. Various doses were used (doses 1-5, with dose 2 being 3 + 1 / 3 times the dose of dose 1, dose 3 being 6 + 2 / 3 times the dose of dose 1, dose 4 being 10 times the dose of dose 1, and dose 5 being 33 + 1 / 3 times the dose of dose 1). The mouse strain and in vitro cell surface copy number of CD19 for each xenograft tumor model are listed as indicated. [Figure 12BC] Figure 1 shows graphs demonstrating in vivo antitumor activity in multiple xenograft models of B-cell malignancies compared with prednisolone or isotype GRM controls. A-C, DB (A), SUPB15 (B), and RS4;11 (C) xenograft models were treated with the indicated agents. Various doses were used (doses 1-5, with dose 2 being 3 + 1 / 3 of dose 1, dose 3 being 6 + 2 / 3 times the dose of dose 1, dose 4 being 10 times the dose of dose 1, and dose 5 being 33 + 1 / 3 times the dose of dose 1). The mouse strain and in vitro cell surface copy number of CD19 for each xenograft tumor model are listed as indicated. [Figure 13A]Antitumor efficacy observed in 10 DLBCL patient-derived xenograft (PDX) models. Germinal center B cell-like (GCB) and non-GCB DLBCL patient-derived PDX models were implanted into NOD-SCID mice and randomized when tumors reached approximately 200 mm3. Mice were treated with a single dose of ADC-1 (dose 3, IP) and tumor growth was monitored compared to control-treated PDXs. Tumor growth inhibition expressed as delta %TGI max = 1 - (treatment group tumor volume on day X - treatment group tumor volume at randomization) / (control tumor volume on day X - control tumor volume at randomization)] * 100. Determined at the time of maximum difference between treatment and control groups. Tumor regression (responder): >105% delta TGI max - responder / no responder. [Figure 13B] Antitumor efficacy observed in 10 DLBCL patient-derived xenograft (PDX) models. Germinal center B cell-like (GCB) and non-GCB DLBCL patient-derived PDX models were implanted in NOD-SCID mice and randomized when tumors reached approximately 200 mm3. Mice were treated with a single dose of ADC-1 (dose 3, IP) and tumor growth was monitored compared to control-treated PDXs. The percent change in tumor volume at the time vehicle control tumors reached 1000 mm3 was calculated by [(tumor volume at time point / tumor volume at randomization) - 1] x 100. [Figure 14A] This graph shows the anti-tumor efficacy observed in an R-CHOP relapsed PDX model. DLBCL patient-derived PDX models from an R-CHOP relapsed patient (who received four cycles of treatment) were implanted into NOD-SCID mice and randomized when tumors reached approximately 200 mm3. Mice were treated with a single dose of ADC-1 or CD19 antibody at dose 3 (IP) and monitored for tumor growth compared to control-treated PDXs. LY-24-0207 is a PDX model derived from a 38-year-old male with GCB DLBCL who relapsed (four cycles) with R-CHOP treatment and showed a poor plateau with ibrutinib. [Figure 14B]This graph shows the anti-tumor efficacy observed in an R-CHOP relapsed PDX model. DLBCL patient-derived PDX models from an R-CHOP relapsed patient (who received four cycles of treatment) were implanted into NOD-SCID mice and randomized when tumors reached approximately 200 mm3. Mice were treated with a single dose of ADC-1 or CD19 antibody at dose 3 (IP), and tumor growth was monitored compared to control-treated PDXs. LY-24-0016 is a PDX model derived from a 59-year-old male with activated B-cell (ABC) DLBCL, who relapsed (four cycles) on R-CHOP treatment and was resistant to ibrutinib. [Figure 15] Figure 1 shows that GRM and ADC-1 induce apoptosis in DLBCL cell lines. Farage, SU-DHL-6, and OCI-LY19 cells were treated with GRM or ADC-1 for the indicated times followed by cell lysis. Lysates were separated by SDS-PAGE and immunoblotted using antibodies against the pro-apoptotic regulator BIM and markers of apoptosis induction (cleaved caspase-3 and cleaved PARP). Arrows indicate apoptotic cleavage products. GAPDH was used as a loading control. [Figure 16] Figure 1 is a graph showing that ADC-1 reduces LC-MS total ion (A280) chromatograms. LC = light chain, LC+2 = light chain + two free thiols, DL(X) = drug linker (number of attachments), HC G0 = heavy chain with G0 glycan. DETAILED DESCRIPTION OF THE INVENTION

[0012] 7. Detailed Description The present disclosure provides antibody drug conjugates (ADCs) comprising a glucocorticoid receptor modulator agonist linked to an anti-CD19 antibody. 7.1.CD19 antibody CD19 is highly expressed in most B-cell malignancies, including DLCB, FL, CLL, and ALL, making it an attractive immunotherapy target for cancers of B-cell origin. We have developed a humanized anti-CD19 antibody that specifically binds to CD19 expressed on the surface of B cells and CD19-positive cancer cells, thereby inhibiting CD19 signaling. In one embodiment, the antibody is composed of two heavy chain variable regions and two light chain variable regions. Each variable region contains three CDRs, enabling the antibody to bind to CD19. A total of six distinct CDRs are present in both variable chains. Furthermore, the antibody contains a human heavy chain constant region, including a human Fc of immunoglobulin class G1 (IgG1). The anti-CD19 antibodies described herein can be fucosylated or defucosylated and exhibit in vitro functionality, immunological safety, and drug-like properties.

[0013] In certain embodiments, the defucosylated antibodies of the present invention have higher affinity for certain FcγR receptors expressed on immune cells. For example, a defucosylated IgG1 antibody exhibits increased binding to FcγRIIIA expressed on NK cells compared to the fucosylated form of the antibody, resulting in enhanced activity in purified natural killer cell or peripheral blood mononuclear cell (PBMC) ADCC assays. The ADCC activity of anti-CD19 antibodies can be demonstrated using ADCC bioassay techniques known in the art. For example, an ADCC reporter bioassay against a human FcγRIIIa V158 or F158 allele variant reporter strain can be used (see Example 3).

[0014] In various embodiments, the antibody comprises all or a portion of an antibody constant region. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In specific embodiments, the anti-CD19 antibody described herein comprises an IgG1. As used herein, the "constant region" of an antibody encompasses native constant regions, allotypes, or variants.

[0015] The light chain constant region of an anti-CD19 antibody can be a light chain kappa (κ) region or a lambda (λ) region. The light chain λ region can be any one of the known subtypes, e.g., λ1, λ2, λ3, or λ4. In some embodiments, the anti-CD19 antibody comprises a light chain kappa (κ) region.

[0016] The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. A monoclonal antibody is derived from a single clone, including any eukaryotic clone.

[0017] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, generally chosen from a human immunoglobulin template.

[0018] "Humanized" forms of non-human (e.g., murine) antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.

[0019] A "human antibody" includes antibodies having the amino acid sequence of a human immunoglobulin and also includes antibodies isolated from a human immunoglobulin library or from an animal that is transgenic for one or more human immunoglobulins and does not express endogenous functional immunoglobulins. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0020] The anti-CD19 antibodies of the present disclosure include full-length (intact) antibody molecules.

[0021] Anti-CD19 antibodies may be antibodies whose sequences have been modified to alter at least one constant region-mediated biological effector function. For example, the anti-CD19 antibodies described herein encompass antibodies that have been modified to acquire or improve at least one constant region-mediated biological effector function, relative to an unmodified antibody, e.g., to enhance FcγR interaction (see, e.g., U.S. Patent Application Publication No. 2006 / 0134709; U.S. Patent No. 8,642,292 B2), or to enhance the ability of the antibody to mediate ADCC. For example, an anti-CD19 antibody of the present disclosure may have a constant region that binds FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB with greater affinity than the corresponding unmodified constant region. Anti-CD19 antibodies of the present disclosure may have an altered Fc region and mediate an enhanced ADCC response, where the ADCC response is enhanced relative to an antibody having the same variable region (i.e., VH and VL) and wild-type IgG1 Fc region (i.e., wild-type CL, CH1, CH2, and CH3). Anti-CD19 antibodies with high affinity for human CD19 may be desirable for therapeutic and diagnostic uses. Accordingly, the present disclosure contemplates antibodies with high binding affinity for human CD19. In certain embodiments, the anti-CD19 antibodies bind to human CD19 with an affinity of about 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or 0.1 nM. In some embodiments, the antibodies bind to human CD19 with an affinity ranging from about 0.1 nM to about 10 nM.

[0022] In some embodiments, the invention provides monoclonal anti-CD19 antibodies that comprise two sets of six different complementarity determining regions (CDRs), two sets of two different variable regions, two complete heavy chains, and two complete light chains.

[0023] In some embodiments, the antibody is a recombinant, defucosylated, humanized, IgG1 kappa monoclonal antibody that binds to human CD19.

[0024] In one embodiment, the antibody comprises six CDRs comprising the following sequences:

[0025] [ka]

[0026] In some embodiments, an antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence set forth as SEQ ID NO: 1, a CDR-H2 having the amino acid sequence set forth as SEQ ID NO: 2, a CDR-H3 having the amino acid sequence set forth as SEQ ID NO: 3, a CDR-L1 having the amino acid sequence set forth as SEQ ID NO: 4, a CDR-L2 having the amino acid sequence set forth as SEQ ID NO: 5, and a CDR-L3 having the amino acid sequence set forth as SEQ ID NO: 6.

[0027] In some embodiments, the antibody of the disclosure has the sequence of SEQ ID NO:7:

[0028] [ka] and a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 8:

[0029] [ka] The light chain variable region comprises:

[0030] In some embodiments, an antibody of the disclosure comprises a heavy chain comprising or consisting of the amino acid sequence set forth as SEQ ID NO:9 (where the constant region is in standard text, the variable heavy domain is underlined, and the CDRs are underlined and bold italicized (disclosed as SEQ ID NOs: 1-3, respectively, in order of appearance), and an N-linked glycosylation site is indicated with a subscript "g"):

[0031] [ka] and a light chain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 10 (the constant region is in standard text, the variable light domain is underlined, and the CDRs are underlined and in bold italics (CDR sequences disclosed as SEQ ID NOs: 4 to 6, respectively, in order of appearance)):

[0032] [ka] Includes:

[0033] In one embodiment, the antibody of the present disclosure comprises or consists of a light chain according to SEQ ID NO: 10 and a heavy chain according to SEQ ID NO: 11, which is the amino acid sequence of SEQ ID NO: 9 with the C-terminal lysine truncated:

[0034] [ka]

[0035] In one embodiment, the antibody is a CD19 mAb having a heavy chain amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 and a light chain amino acid sequence according to SEQ ID NO:10.

[0036] In one embodiment, the antibody heavy chain of the present disclosure is encoded by the following nucleotide sequence (full length sequence disclosed as SEQ ID NO: 12):

[0037] [ka]

[0038] The secretory signal peptide is in italics, including the final stop codon (TGA), the constant region is in bold, and the CDRs are underlined.

[0039] In one embodiment, the light chain of an antibody of the present disclosure is encoded by the following nucleotide sequence (full length sequence disclosed as SEQ ID NO: 13):

[0040] [ka]

[0041] The secretory signal peptide is in italics, including the final stop codon (TGA), the constant region is in bold, and the CDRs are underlined.

[0042] In some embodiments, the antibody comprises a human heavy chain constant region comprising a human CH1, human hinge, human CH2, and human CH3 domains. In some embodiments, the encoded heavy chain constant region comprises an Fc portion, wherein the Fc portion is a human IgG1, IgG2, IgG3, IgG4, or IgM isotype. In certain embodiments, the Fc is IgG1 and the allotype is z, not a. In certain embodiments, the light chain is a kappa light chain.

[0043] In some embodiments, the antibody comprises a defucosylated IgG1 Fc constant region. Defucosylation can be performed by techniques known in the art. See, e.g., Mol Cancer Ther (2020) 19(5):1102-1109 and PNAS (2013) 110(14) 5404-5409. For example, production of antibodies in cell lines defective in GDP-fucose formation, e.g., due to a deficiency in GDP-mannose 4,6-dehydratase; production of antibodies in cells with reduced levels of fucosyltransferase; production of antibodies in cells with reduced levels of GDP-fucose transporter; production of antibodies in cells overexpressing β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (GnT-III); or production of antibodies in cells expressing bacterial GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In certain embodiments, the cells used to produce the defucosylated anti-CD19 antibodies of the invention are CHO cells engineered to express Pseudomonas RMD. The degree of defucosylation of the antibody can be determined by techniques known in the art. Generally, the antibody is 70% or more, 80% or more, 90% or more, or about 99% or about 100% defucosylated. Preferably, the degree of defucosylation is 90% or more. In some embodiments, the antibody is 70% or more, 80% or more, 90% or more, or about 100% defucosylated at position ASN-303 (EU:ASN-297) of SEQ ID NO:9 or SEQ ID NO:11. Defucosylation can be determined by a hydrophilic interaction chromatography (HILIC) assay technique, in which the degree of defucosylation is determined by polarity-dependent separation of fragmented antibodies.

[0044] In one embodiment, the total defucosylated glycan species is determined by analyzing the released N-linked glycans by HILIC with fluorescence detection. The glycans are released using peptide N-glycosidase F (PNGaseF) and then labeled with a fluorescent tag. The fluorescently labeled N-linked glycans are analyzed by HILIC with fluorescence detection. The percent defucosylated glycan species is determined based on the sum of the peak areas of all defucosylated glycan peaks relative to the total peak area of ​​all glycan peaks in the chromatogram. All peaks with a relative abundance of 0.5% or greater are included in the determination of the percent defucosylated glycan species.

[0045] The present disclosure encompasses polynucleotide molecules encoding immunoglobulin light and heavy chain genes of anti-CD19 antibodies, vectors containing such polynucleotides, and host cells capable of producing the anti-CD19 antibodies of the present disclosure.

[0046] The anti-CD19 antibodies of the present disclosure can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To express the antibody recombinantly, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cells and, optionally, secreted into the medium in which the host cells are cultured, from which the antibody can be recovered.

[0047] To generate polynucleotides encoding such anti-CD19 antibodies, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNAs encoding the light and heavy chain variable sequences, for example, using the polymerase chain reaction (PCR).

[0048] Once DNA fragments encoding the VH and VL segments relevant to an anti-CD19 antibody are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0049] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, CH3, and optionally CH4). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but in certain embodiments, is IgG1 or IgG4. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0050] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region, but in certain embodiments is a kappa constant region.

[0051] To express the anti-CD19 antibody of the present disclosure, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, but more commonly, both genes are inserted into the same expression vector.

[0052] The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present). The expression vector may already contain antibody constant region sequences prior to insertion of the light or heavy chain sequences associated with the anti-CD19 antibody. For example, one approach to converting the VH and VL sequences associated with an anti-CD19 monoclonal antibody into full-length antibody genes is to insert those VH and VL sequences into an expression vector already encoding heavy and light chain constant regions, respectively, such that the VH segment is operably linked to a CH segment within the vector and the VL segment is operably linked to a CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

[0053] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.

[0054] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0055] In certain embodiments, antibody expression is carried out in eukaryotic cells, such as mammalian host cells, that optimally secrete properly folded, immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selection marker, as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect host cells with DNA encoding either the light chain or the heavy chain (but not both) of an anti-CD19 antibody of the present disclosure.

[0056] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to human CD19, and molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0057] For recombinant expression of anti-CD19 antibodies of the present disclosure, host cells can be co-transfected with two expression vectors of the present disclosure: a first vector encoding heavy chain-derived polypeptides and a second vector encoding light chain-derived polypeptides. The two vectors can contain identical selectable markers, or each can contain a separate selectable marker. Alternatively, a single vector can be used that encodes both heavy and light chain polypeptides.

[0058] 7.2. Anti-CD19 Antibodies Linked to Glucocorticoid Receptor Modulators Provided herein are antibody drug conjugates (ADCs) comprising a glucocorticoid receptor modulator (GRM) agonist linked to an anti-CD19 antibody.

[0059] In certain embodiments, a composition comprising: (a) an anti-CD19 antibody; and (b) an antibody of Formula (I):

[0060] [ka] (In the formula, R 1 is hydrogen or -P(=O)(OH)2), Radical glucocorticoid receptor modulators 1. An antibody drug conjugate comprising: Additionally, antibodies and glucocorticoid receptor modulators may be used in combination with a compound of the formula: where R is the point of attachment of the antibody to the linker via a cysteine ​​residue in the antibody, resulting in an -S- group upon linkage; AA1 and AA2 are alanine (Ala); and p and q are 1. is conjugated by a linker of Antibody drug conjugates are provided.

[0061] In certain embodiments, a compound of formula (II):

[0062] [ka] where A is an anti-CD19 antibody, R is the point of attachment of the antibody to the linker via a cysteine ​​residue of the antibody, resulting in an -S- group when linked, AA1 and AA2 are alanine (Ala), and R 1 is hydrogen or -P(=O)(OH)2, and n is 2 to 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 2, 3, 4, 5, 6, 7, or 8. In other embodiments, n is 1, 2, 3, 4, or 5. In other embodiments, n is 2, 3, 4, or 5. In some embodiments, n is 2, 4, 6, or 8. In other embodiments, n is 2, 4, or 6. In other embodiments, n is 1. In other embodiments, n is 2. In other embodiments, n is 3. In other embodiments, n is 4. In other embodiments, n is 5. In other embodiments, n is 6. In other embodiments, n is 7. In another embodiment, n is 8.

[0063] In certain embodiments, the anti-CD19 antibody of the ADC comprises the complementarity-determining regions (CDRs) set forth as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In certain embodiments, the anti-CD19 antibody comprises the heavy chain variable region set forth as SEQ ID NO:7 and the light chain variable region set forth as SEQ ID NO:8. In certain embodiments, the anti-CD19 antibody comprises the heavy chain set forth as SEQ ID NO:9 or SEQ ID NO:11 and the light chain set forth as SEQ ID NO:10, where n is predominantly 2, 4, and 6. In certain embodiments, the ADC is ADC-1 and the antibody is a CD19 mAb. In certain embodiments, the anti-CD19 antibody heavy chain is encoded by the nucleotide sequence set forth as SEQ ID NO:12, and the light chain is encoded by the nucleotide sequence set forth as SEQ ID NO:13.

[0064] 7.3. Composition The anti-CD19 GRM agonist ADCs of the present disclosure may be provided as compositions suitable for administration to a subject. In some embodiments, the anti-CD19 GRM agonist ADC composition is a pharmaceutical composition comprising an anti-CD19 GRM agonist ADC of the present disclosure and a pharmaceutically acceptable carrier.

[0065] An ADC composition can include a mixture of ADCs having different integer values ​​of n according to structural formula II. The number of GRM agonist drugs linked to an ADC depends on the number of available attachment sites on the antibody. "Drug-antibody ratio" or "DAR," as used herein, refers to the molar ratio of GRM drug to antibody in an ADC composition.

[0066] In embodiments, the formulation consists essentially of a mixture of ADCs according to structural formula II having n values ​​of 2, 4, and 6 and DARs between 2 and 6.

[0067] 7.4.How to use In certain embodiments, the methods described herein involve treating a patient with a B-cell malignancy, such as diffuse large B-cell lymphoma (DLBCL), acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL), with a CD19 GRM agonist ADC. In some embodiments, a composition comprising a CD19 GRM agonist ADC is administered to a subject in need thereof. In some embodiments, the method involves administering an ADC that results in delivery of a compound of Formula III, a compound of Formula IV, or both compounds of Formulas III and IV to the subject. In some embodiments, a compound of Formula III, a compound of Formula IV, or both compounds of Formulas III and IV is delivered to the B-cell malignancy in the subject.

[0068] When delivered as single agents, CD19-GRM agonist ADCs inhibit the growth of subcutaneous xenografts of human tumor cell lines derived from B-cell malignancies (see examples for diffuse large B-cell lymphoma (DLBCL: OCI-LY19 and SU-DHL-6; see Figures 8 and 10) and acute lymphoblastic leukemia (ALL: RS4; 11; see Figure 9).

[0069] 7.5. Process for Producing Anti-CD19 ADCs In certain embodiments, described herein are processes for making anti-CD19-GRM agonist ADCs. In certain embodiments, described herein are processes for making anti-CD19-GRM agonist ADCs, wherein the antibody is an anti-CD19 antibody comprising two heavy chains, each consisting of the amino acid sequence set forth as SEQ ID NO:9 or SEQ ID NO:11, and two light chains, each consisting of the amino acid sequence set forth as SEQ ID NO:10, and the antibody is defucosylated at position ASN-303 (EU:ASN-297) of SEQ ID NO:9 or SEQ ID NO:11, and the process comprises reducing the antibody with an excess of a reducing agent, followed by conjugating the antibody to an excess of a drug linker of Formula V.

[0070] 8. Exemplary Embodiments Various specific embodiments have been illustrated and described, some of which are set forth below, but it will be understood that various changes can be made without departing from the spirit and scope of the invention.

[0071] 1. The following structure:

[0072] [ka] (In the formula, A is an antibody, R is the point of attachment of the antibody via a cysteine ​​residue on the antibody, which upon linkage provides an -S- group; AA1 and AA2 are alanine (Ala), R 1 is hydrogen or -P(=O)(OH)2, n is an integer from 1 to 10. 1. An anti-CD19 antibody drug conjugate comprising: The antibody, a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and a light chain variable region comprising the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain an anti-CD19 antibody comprising CDR-H1 comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1), CDR-H2 comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2), CDR-H3 comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3), CDR-L1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4), CDR-L2 comprises the amino acid sequence WASTRHT (SEQ ID NO: 5), and An anti-CD19 antibody drug conjugate, wherein CDR-L3 comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6).

[0073] 2. The anti-CD19 antibody drug conjugate of embodiment 1, wherein the antibody is an IgG antibody.

[0074] 3. The anti-CD19 antibody drug conjugate of embodiment 2, wherein the antibody is an IgG1 antibody.

[0075] 4. The anti-CD19 antibody drug conjugate of embodiment 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO:8.

[0076] 5. The anti-CD19 antibody drug conjugate of embodiment 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth as SEQ ID NO:9 or SEQ ID NO:11, and a light chain comprising the amino acid sequence set forth as SEQ ID NO:10.

[0077] 6. The anti-CD19 antibody drug conjugate of embodiment 1, wherein the antibody consists of two identical heavy chains and two identical light chains, each heavy chain consisting of the amino acid sequence set forth as SEQ ID NO:9 or SEQ ID NO:11, and each light chain consisting of the amino acid sequence set forth as SEQ ID NO:10.

[0078] 7. The anti-CD19 antibody drug conjugate according to any one of embodiments 1 to 6, wherein the antibody is defucosylated.

[0079] 8. The anti-CD19 antibody drug conjugate according to embodiment 7, wherein the antibody is more than 70%, more than 80%, more than 90%, or about 99% or about 100% defucosylated.

[0080] 9. The anti-CD19 antibody drug conjugate according to embodiment 7 or embodiment 8, wherein the defucosylation is at position ASN-303 (EU:ASN-297) of SEQ ID NO: 9 or SEQ ID NO: 11.

[0081] 10. A pharmaceutical composition comprising the anti-CD19 antibody-drug conjugate of any one of embodiments 1 to 9 and a pharmaceutically acceptable carrier.

[0082] 11. A method of treating a B-cell malignancy, comprising administering to a patient in need thereof an antibody drug conjugate according to any one of embodiments 1-9.

[0083] 12. The anti-CD19 antibody drug conjugate of any one of embodiments 1 to 9, wherein n is 2, 4 or 6.

[0084] 13. The anti-CD19 antibody drug conjugate of any one of embodiments 1 to 9, wherein n is 2.

[0085] 14. The anti-CD19 antibody drug conjugate of any one of embodiments 1 to 9, wherein n is 4.

[0086] 15. The anti-CD19 antibody drug conjugate of any one of embodiments 1 to 9, wherein n is 6.

[0087] 16. A heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and a light chain variable region comprising the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain an anti-CD19 antibody comprising: CDR-H1 comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1), CDR-H2 comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2), CDR-H3 comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3), CDR-L1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4), CDR-L2 comprises the amino acid sequence WASTRHT (SEQ ID NO: 5), and CDR-L3 comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6), Anti-CD19 antibody.

[0088] 17. The anti-CD19 antibody of embodiment 16, which is an IgG antibody.

[0089] 18. The anti-CD19 antibody of embodiment 17, which is an IgG1 antibody.

[0090] 19. The anti-CD19 antibody according to embodiment 16, comprising a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO:8.

[0091] 20. The anti-CD19 antibody according to embodiment 16, comprising a heavy chain comprising the amino acid sequence set forth as SEQ ID NO:9 or SEQ ID NO:11, and a light chain comprising the amino acid sequence set forth as SEQ ID NO:10.

[0092] 21. The anti-CD19 antibody according to any one of embodiments 16 to 20, which is defucosylated.

[0093] 22. The anti-CD19 antibody according to embodiment 21, which is at least 70%, at least 80%, at least 90%, or about 99% or about 100% defucosylated.

[0094] 23. The anti-CD19 antibody according to embodiment 21 or embodiment 22, wherein the defucosylation is at position ASN-303 (EU:ASN-297) of SEQ ID NO: 9 or SEQ ID NO: 11.

[0095] 24. The anti-CD19 antibody according to embodiment 16 or 19, which is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, a humanized antibody, a single-chain antibody, a single-domain antibody, a camelized antibody, an scFv-Fc antibody, Fab, Fab', (Fab')2, Fv or scFv.

[0096] 25. The anti-CD19 antibody according to embodiment 24, which is defucosylated.

[0097] 26. The anti-CD19 antibody according to embodiment 25, which is at least 70%, at least 80%, at least 90%, or about 99% or about 100% defucosylated.

[0098] 27. The anti-CD19 antibody according to embodiment 25 or embodiment 26, wherein the defucosylation is at position ASN-303 (EU:ASN-297) of SEQ ID NO: 9 or SEQ ID NO: 11.

[0099] 28. The following structure:

[0100] [ka] (In the formula, A is an antibody, R is the point of attachment of the antibody via a cysteine ​​residue on the antibody, which upon linkage provides an -S- group; AA1 and AA2 are alanine (Ala), R 1 is -P(=O)(OH)2, n is an integer from 2 to 8. 1. An anti-CD19 antibody drug conjugate comprising: The antibody, Two heavy chains each consisting of the amino acid sequence set forth as SEQ ID NO: 9 or SEQ ID NO: 11 and two light chains each consisting of the amino acid sequence set forth as SEQ ID NO: 10 an anti-CD19 antibody comprising the antibody is defucosylated at position ASN-303 (EU:ASN-297) of SEQ ID NO: 9 or SEQ ID NO: 11; Anti-CD19 antibody drug conjugate.

[0101] 29. The anti-CD19 antibody according to embodiment 28, wherein n is 2.

[0102] 30. The anti-CD19 antibody according to embodiment 28, wherein n is 4.

[0103] 31. The anti-CD19 antibody according to embodiment 28, wherein n is 6.

[0104] 32. The anti-CD19 antibody according to embodiment 28, wherein n is 8.

[0105] 33. One or more polynucleotides encoding the anti-CD19 antibody according to embodiment 16.

[0106] 34. The following structure:

[0107] [ka] (In the formula, A is an antibody, R is the point of attachment of the antibody via a cysteine ​​residue on the antibody, which upon linkage provides an -S- group; AA1 and AA2 are alanine (Ala), and R 1 is -P(=O)(OH)2, n is an integer from 2 to 8. 1. A process for making an anti-CD19 antibody drug conjugate comprising: The antibody, It comprises two heavy chains each consisting of the amino acid sequence set forth as SEQ ID NO:9 or SEQ ID NO:11 and two light chains each consisting of the amino acid sequence set forth as SEQ ID NO:10. an anti-CD19 antibody, the antibody is defucosylated at position ASN-303 (EU:ASN-297) of SEQ ID NO: 9 or SEQ ID NO: 11; The process involves reducing the antibody with an excess of a reducing agent, after which the antibody is treated with an excess of a compound having the following structure:

[0108] [ka] and conjugating the compound to a drug linker comprising: process. [Example]

[0109] 9. Working Example The following examples, which highlight certain features and properties of exemplary embodiments of the antibodies and binding fragments described herein, are presented for illustrative purposes.

[0110] 9.1 Example 1: Generation of CD19 antibody A CD19 antibody consisting of the complementarity-determining regions (CDRs) of the murine antibody clone 4B12 (murine anti-human CD19) was isolated using hybridoma technology. These CDR sequences (SEQ ID NOS: 1-6) were cloned into human IgG1 heavy chain and human kappa light chain constant region domains with nine murine variable heavy chain framework back mutations and two murine variable light chain framework back mutations to generate the heavy chain sequence (SEQ ID NOS: 9) and the light chain sequence (SEQ ID NOS: 10). The humanized CD19 antibody (CD19 mAb) retains the CD19-binding potency and selectivity of the parent murine hybridoma clone 4B12.

[0111] Chinese hamster ovary (CHO) cells were transfected by electroporation with an expression vector (pCD-CD19 antibody) designed to express the CD19 antibody. The cells were grown in a chemically defined medium, which allowed for the selection of cells expressing DHFR and the CD19 antibody from the expression vector. The cells were grown and screened for clonal expansion for anti-CD19 antibody expression. Based on the screening, stable transfectants were identified as parental cell lines for further development.

[0112] This stable transfectant was then transfected by electroporation with an additional vector (pYH9neo-RMD, expressing the Pseudomonas enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD)). This expression vector allows for the production of defucosylated CD19 antibody (greater than 90% defucosylated) in cell lines defective in GDP-fucose formation, e.g., due to a deficiency in GDP-mannose 4,6-dehydratase. Clonal cells were screened for growth and expression of the defucosylated CD19 antibody. Final subclones were selected to generate a master cell bank.

[0113] 9.2 Example 2: Synthesis of ADC-1, a CD19 GRM agonist ADC

[0114] Overview Conjugating the CD19 GRM agonist ADC to the anti-CD19 antibody of Example 1 (R 1 is a phosphate group) and a linker drug containing a GRM of formula I. Formula (III):

[0115] [ka] The phosphate-GRM payload of Formula III is conjugated to the CD19 antibody via an alanine-alanine cleavable linker. Upon administration of the ADC, the phosphate-GRM payload of Formula III is converted to the phosphate-GRM payload of Formula IV:

[0116] [ka] is converted to GRM.

[0117] The conjugated material is processed using hydrophobic interaction chromatography (HIC) to substantially deplete antibodies that do not contain a conjugated linker drug and ADCs that contain 8 GRM molecules per antibody, resulting in a final drug product that contains a mixture of ADCs that contain primarily 2, 4, or 6 GRM molecules and has a DAR of 2-6.

[0118] The analytical procedures used to generate the CD19 GRM agonist ADC, ADC-1, are detailed below.

[0119] 9.2.2. Small Molecule Analysis Procedures Unless otherwise specified, 1 H and 13All C NMR (nuclear magnetic resonance) data were collected on a Varian Mercury Plus 400 MHz, Agilent MR 400 MHz, or Bruker AVIII 300 MHz, 400 MHz, 500 MHz, or 600 MHz instrument. Chemical shifts are quoted in parts per million (ppm). High-performance liquid chromatography (HPLC) and LCMS analytical data are detailed within the experiment or with reference to the conditions listed in Table 1.

[0120] [Table 1]

[0121] 9.2.3. Synthesis of precursor molecules

[0122] 9.2.3.1. Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (GRM of Formula IV)

[0123] [ka]

[0124] Step 1: Synthesis of 4-(bromomethyl)benzaldehyde. Diisobutylaluminum hydride (153 mL, 153 mmol, 1 M in toluene) was added dropwise over 1 h to a solution of 4-(bromomethyl)benzonitrile (20 g, 102 mmol) in toluene (400 mL) at 0 °C. Two additional reactions were set up as above. All three reaction mixtures were combined for purification. The combined reaction mixture was quenched with 10% aqueous HCl (1.5 L), extracted with dichloromethane (3 × 500 mL), dried (NaSO), and the solvent removed under reduced pressure. Purification by column chromatography (silica gel) eluting with 10:1 petroleum ether / ethyl acetate afforded the title compound (50 g, 82% yield). 1 H NMR (400MHz, CDCl3) δ 10.02 (s, 1H), 7.91 - 7.82 (m, 2H), 7.56 (d, J=7.9 Hz, 2H), 4.55 - 4.45 (m, 2H).

[0125] Step 2: Synthesis of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. To a solution of 3-bromoaniline (40 g, 233 mmol) in 1,4-dioxane (480 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (94 g, 372 mmol), potassium acetate (45.6 g, 465 mmol), 2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl (8.07 g, 13.95 mmol), and tris(dibenzylideneacetone)dipalladium(0) (8.52 g, 9.30 mmol). The resulting mixture was then heated at 80 °C under nitrogen for 4 h. An additional reaction was set up as described above. The two reaction mixtures were combined and the solvent was removed under reduced pressure. Purification by column chromatography (silica gel) eluting with 10:1 petroleum ether / ethyl acetate gave the title compound (60 g, 55.4% yield). 1H NMR (400MHz, CDCl3) δ 7.23 - 7.13 (m, 3H), 6.80 (d, J=7.5 Hz, 1H), 3.82 - 3.38 (m, 2H), 1.34 (s, 12H).

[0126] Step 3: Synthesis of tert-butyl (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate. 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (30 g, 137 mmol) and di-tert-butyl dicarbonate (38.9 g, 178 mmol) were mixed in toluene (600 mL) at 100° C. for 24 hours. Another reaction was set up as described above. The two reaction mixtures were combined and the solvent was removed under reduced pressure. The crude residue was dissolved in ethyl acetate (1.5 L) and washed with 0.1 N aqueous HCl (3×2 L), brine (3 L), dried (NaSO), filtered, and concentrated under reduced pressure to give the title compound (50 g, 57% yield). 1 H NMR (400MHz, CDCl3) δ 7.63 (br m, 2H), 7.48 (d, J=7.1 Hz, 1H), 7.37 - 7.28 (m, 1H), 1.52 (s, 9H), 1.34 (s, 12H).

[0127] Step 4: Synthesis of tert-butyl (3-(4-formylbenzyl)phenyl)carbamate. A mixture of 4-(bromomethyl)benzaldehyde (24.94 g, 125 mmol), 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) dichloromethane (13.75 g, 18.80 mmol), tert-butyl (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (20 g, 62.7 mmol), and potassium carbonate (43.3 g, 313 mmol) in tetrahydrofuran (400 mL) was heated to 80° C. for 12 hours. Another additional reaction was set up as described above. The two reaction mixtures were combined, diluted with water (500 mL), and extracted with ethyl acetate (3×500 mL). The combined organic layers were dried (Na2SO4) and the solvent was removed under reduced pressure. Purification by column chromatography (silica gel) eluting with 10:1 petroleum ether / ethyl acetate gave the title compound (15 g, 38.4% yield). 1 H NMR (400MHz, CDCl3) δ 9.95 (s, 1H), 7.78 (d, J=7.9 Hz, 2H), 7.33 (d, J=7.9 Hz, 2H), 7.27 - 7.13 (m, 3H), 6.82 (d, J=7.1 Hz, 1H), 6.47 (br. s., 1H), 4.00 (s, 2H), 1.48 (s, 9H).

[0128] Step 5: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one. (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (20 g, 44.2 mmol) was suspended in 440 mL of 40% aqueous HBF solution, and the mixture was stirred at 25 °C for 48 h. After completion of the reaction, 2 L of water was added, and the solid was collected by filtration. The solid was washed with water (1 L) and then with methanol (200 mL) to give the title compound (11 g, 60.3% yield). 1 H NMR (400MHz, DMSO-d6) δ 7.25 (d, J=10.1 Hz, 1H), 6.28 (d, J=10.1 Hz, 1H), 6.10 (s, 1H), 5.73 - 5.50 (m, 1H), 5.39 (br. s., 1H), 4.85 - 4.60 (m, 2H), 4.50 (d, J=19.4 Hz, 1H), 4.20 - 4.04 (m, 2H), 2.46 - 2.06 (m, 6H), 1.87 - 1.75 (m, 1H), 1.56 - 1.30 (m, 6H), 0.83 (s, 3H).

[0129] Step 6: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one. A suspension of (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (4.4 g, 10.67 mmol) and MgSO (6.42 g, 53.3 mmol) in acetonitrile (100 mL) was stirred for 1 h at 20 ° C. A solution of tert-butyl (3-(4-formylbenzyl)phenyl)carbamate (3.65 g, 11.74 mmol) in acetonitrile (100 mL) was added in one portion. Trifluoromethanesulfonic acid (9.01 mL, 53.3 mmol) was added dropwise while maintaining the internal temperature below room temperature using an ice bath. After the addition, the mixture was stirred at 20° C. for 2 hours. Three additional reactions were set up as above. All four reaction mixtures were combined and the solvent was removed under reduced pressure. Purification by preparative HPLC gave the title compound (4.5 g, 14.2% yield). LCMS (Method a, Table 1) R t =2.65 min;MS m / z=606.2(M+H) + . 1H NMR (400 MHz, DMSO) δ 7.34 (d, J = 7.7 Hz, 2H), 7.24 (dd, J = 16.2, 9.0 Hz, 3H), 6.88 (t, J = 7.8 Hz, 1H), 6.43 - 6.33 (m, 3H), 6.30 (d, J = 10.2 Hz, 1H), 6.13 (s, 1H), 5.75 - 5.55 (m, 1H), 5.52 (d, J = 4.1 Hz, 1H), 5.44 (s, 1H), 5.11 (t, J = 6.0 Hz, 1H), 4.94 (s, 3H), 4.51 (dd, J = 19.6, 6.5 Hz, 1H), 4.29 - 4.07 (m, 2H), 3.73 (s, 2H), 2.76 - 2.55 (m, 1H), 2.39 - 2.13 (m, 2H), 2.10 - 1.96 (m, 1H), 1.85 - 1.61 (m, 3H), 1.61 - 1.39 (m, 4H), 0.86 (s, 3H). Preparative HPLC method: Instrument: Gilson 281 semi-preparative HPLC system; Mobile phase: A: formic acid / HO = 0.01 v / v%; B: acetonitrile; Column: Luna C18 150*25 5 micron; Flow rate: 25 mL / min; Monitoring wavelength: 220 nm and 254 nm.

[0130] [Table 2]

[0131] 9.2.3.2. Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate (phosphate-GRM of Formula III)

[0132] [ka]

[0133] Diphosphoryl chloride (6.86 g, 27.2 mmol) was dissolved in tetrahydrofuran (30 mL) to prepare a solution of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a- To a solution of dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (3.30 g, 5.45 mmol) and para-toluenesulfonic acid (0.938 g, 5.45 mmol) was added dropwise at −40°C. The mixture was stirred at −40°C for 2 hours. Three additional reactions were set up as described above. The combined reaction mixture was quenched with water (450 mL) at −40°C, warmed to 0°C, and the pH was adjusted to approximately 7 by adding saturated aqueous NaHCO3. The suspension was filtered at a pH of approximately 6 as a large liquid volume filled the flask. The filtrate was then adjusted to pH 7 and rinsed through the filtered solids. The filtrate was acidified back to pH 3 by adding 1 M aqueous HCl (200 mL), resulting in a suspension. The mixture was filtered, and the combined solid was rinsed twice with the mother liquor and collected. The crude solid was dried under vacuum and purified by preparative HPLC on a Nano-micro Kromasil C18 3-micron column (100 x 40 mm). A gradient of acetonitrile (A) and 1% trifluoroacetic acid in water (B) was used at a flow rate of 50 mL / min (0-5.0 min, 20% A, 5.0-25.0 min, linear gradient 20-50% A, 5 min hold). The pH of the purified fractions was adjusted to approximately 5 and lyophilized to give the title compound as a white solid (3.50 g, 23% yield). LCMS (Method b, Table 1) R t =0.707 min, m / z 686.2(M+H + ). 1H NMR (400 MHz, DMSO-d6) δ 7.35 (d, J = 7.9 Hz, 2H), 7.26 (td, J = 7.3, 6.2, 3.1 Hz, 3H), 7.07 (t, J = 7.9 Hz, 1H), 6.73 - 6.60 (m, 3H), 6.30 (dd, J = 10.2, 1.9 Hz, 1H), 6.12 (d, J = 2.4 Hz, 1H), 5.75 - 5.55 (m, 1H), 5.54 (s, 1H), 4.98 - 4.86 (m, 2H), 4.59 (dd, J = 18.2, 8.3Hz, 1H), 4.25 - 4.17 (m, 1H), 3.83 (s, 2H), 2.79 - 2.56 (m, 1H), 2.37 - 2.17 (m, 2H), 2.05 (dt, J = 13.7, 3.6 Hz, 1H), 1.72 (q, J = 9.3, 8.1 Hz, 3H), 1.60 - 1.41 (m, 1H), 1.50 (s, 3H), 0.88 (s, 3H). 19 F NMR (376 MHz, DMSO-d6) δ -73.76, -164.99, -186.36. 31 P NMR (162 MHz, DMSO-d6) δ -1.17.

[0134] 9.2.3.3. Synthesis of (S)-2-(2-bromoacetamido)-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide (Drug Linker of Formula VI)

[0135] [ka]

[0136] Step 1: Synthesis of tert-butyl ((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate.

[0137] 2,6-Lutidine (0.5 mL, 4.29 mmol) was dissolved in tetrahydrofuran (5.5 mL) and the resulting solution was (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7 To a room temperature suspension of 8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (0.505 g, 0.834 mmol), (S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid [CAS number 27317-69-7, purchased from Chem-Impex International, Inc.] (0.217 g, 0.835 mmol), and HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (0.481 g, 1.266 mmol). After 16 h, the reaction was diluted with EtOAc (40 mL), washed with 1N aqueous HCl (2 × 20 mL), saturated aqueous NaHCO (20 mL), brine (20 mL), dried (NaSO), filtered, and the solvent removed under reduced pressure. Purification by chromatography (silica, 40 g) eluting with a gradient of 0–10% MeOH / DCM afforded the title compound as an off-white solid (0.648 g, 92% yield). LCMS (Method c, ESI, Table 1) R t =0.89 min, m / z=848.6(M+H + ). 1H NMR (600 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.94 (d, J = 7.3 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.38 (t, J = 2.0 Hz, 1H), 7.38 - 7.33 (m, 2H), 7.26 (dd, J = 10.2, 1.5 Hz, 1H), 7.26 - 7.22 (m, 2H), 7.19 (t, J = 7.9 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 6.91 (dt, J = 7.7, 1.2 Hz, 1H), 6.29 (dd, J = 10.1, 1.9 Hz, 1H), 6.14 - 6.11 (m, 1H), 5.72 - 5.56 (m, 1H), 5.53 - 5.49 (m, 1H), 5.44 (s, 1H), 5.09 (t, J = 5.9 Hz, 1H), 4.94 (d, J = 5.2 Hz, 1H), 4.51 (dd, J = 19.4, 6.1 Hz, 1H), 4.35 (t, J = 7.1 Hz, 1H), 4.23 - 4.16 (m, 2H), 3.97 (t, J = 7.2 Hz, 1H), 3.88 (s, 2H), 2.65 (ddt, J = 28.2, 15.3, 7.9 Hz, 1H), 2.36 - 2.16 (m, 2H), 2.07 - 2.01 (m, 1H), 1.78 - 1.63 (m, 3H), 1.57 - 1.50 (m, 1H), 1.49 (s, 3H), 1.36 (s, 9H), 1.26 (d, J = 7.1 Hz, 3H), 1.20 - 1.15 (m, 3H), 0.86 (s, 3H).

[0138] Step 2: Synthesis of (S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide Trifluoroacetic acid (2.0 mL, 26.0 mmol) was dissolved in tert-butyl ((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b, To a room temperature solution of 7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (0.648 g, 0.764 mmol) was added. The reaction was stirred in an open flask for 20 minutes, and then the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (60 mL), washed with saturated aqueous NaHCO (3 x 20 mL), saturated aqueous NaBr (20 mL), dried (NaSO), filtered, and the solvent was removed under reduced pressure. The residue was triturated with MTBE / heptane and the solvent was removed to give the title compound, which was used in the next step without further purification. LCMS (Method c, ESI, Table 1)R t =0.72min;m / z748.5(M+H + ).

[0139] Step 3: Synthesis of (S)-2-(2-bromoacetamido)-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide.

[0140] 2,6-Lutidine (0.2 mL, 1.717 mmol) was dissolved in dimethylformamide (4 mL) to prepare (S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8 To a room temperature solution of a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide (0.286 g, 0.382 mmol) and bromoacetic acid N-hydroxysuccinimide ester (0.414 g, 1.753 mmol) was added. The reaction was complete within 20 minutes by LCMS. A second reaction of the same size was set up. Both reactions were combined for purification by preparative HPLC on a Phenomenex C18(2) 10 micron column (250 x 50 mm). A gradient of acetonitrile (A) and 1% trifluoroacetic acid in water (B) was used at a flow rate of 90 mL / min (0-5.0 min, 15% A, 5.0-20.0 min, linear gradient 15-95% A, 5 min hold). The product fractions were freeze-dried to give the title compound as a white solid (0.254 g, 38% yield). LCMS (Method c, ESI, Table 1) t =0.82 min, m / z868.4, 870.3(M+H) + .1 H NMR (600 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.45 (d, J = 7.3 Hz, 1H), 8.20 (d, J = 7.2 Hz, 1H), 7.46 (dd, J = 8.4, 2.2 Hz, 1H), 7.40 (t, J = 1.9 Hz, 1H), 7.38 - 7.33 (m, 2H), 7.28 - 7.19 (m, 4H), 7.19 (d, J = 7.9 Hz, 1H), 6.91 (dt, J = 7.7, 1.3 Hz, 1H), 6.29 (dd, J = 10.2, 1.9 Hz, 1H), 6.12 (d, J = 2.0 Hz, 1H), 5.64 (dddd, J = 48.5, 11.4, 6.8, 1.9 Hz, 1H), 5.51 (d, J = 4.4 Hz, 1H), 5.45 (s, 1H), 4.94 (d, J = 5.3 Hz, 1H), 4.51 (d, J = 19.4 Hz, 1H), 4.40 - 4.33 (m, 1H), 4.31 (p, J = 7.2 Hz, 1H), 4.23 - 4.16 (m, 2H), 3.94 - 3.87 (m, 4H), 2.71 - 2.58 (m, 1H), 2.27 (ddq, J = 31.2, 12.4, 6.3, 5.7 Hz, 1H), 2.04 (dt, J = 13.7, 3.7 Hz, 1H), 1.77 - 1.63 (m, 3H), 1.57 - 1.50 (m, 1H), 1.50 (s, 3H), 1.27 (d, J = 7.1 Hz, 3H), 1.21 (d, J = 7.1 Hz, 3H), 0.86 (s, 3H).

[0141] 9.2.3.4. Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate (Drug Linker of Formula V)

[0142] [ka]

[0143] Step 1: Synthesis of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate.

[0144] 2,6-Lutidine (1.2 mL, 10.30 mmol) was dissolved in tetrahydrofuran (15 mL) and the resulting solution was (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6 To a room temperature solution of b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (1.377 g, 2.27 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine [CAS number 87512-31-0, purchased from Chem-Impex International, Inc.] (0.878 g, 2.295 mmol), and HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (1.327 g, 3.49 mmol). After 15 h, the mixture was diluted with EtOAc (100 mL), washed with 1N aqueous HCl (2 × 50 mL), saturated aqueous NaHCO (50 mL), brine (50 mL), dried (NaSO), filtered, and the solvent removed under reduced pressure. Purification by chromatography (silica, 120 g) eluting with a gradient of 0-10% MeOH / DCM gave the product as a white solid (1.98 g, 90% yield). LCMS (Method c, APCI, Table 1) yielded 1.98 g (90% yield). t =0.98 min, m / z970.6(M+H + ). 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.06 (d, J = 7.3 Hz, 1H), 7.91 - 7.84 (m, 2H), 7.71 (t, J = 7.8 Hz, 2H), 7.53 (d, J = 7.4 Hz, 1H), 7.49 - 7.13 (m, 12H), 6.91 (dt, J = 7.7, 1.4 Hz, 1H), 6.29 (dd, J = 10.2, 1.9 Hz, 1H), 6.14 (q, J = 1.6 Hz, 1H), 5.73 - 5.54 (m, 1H), 5.52 (dd, J = 4.4, 1.8 Hz, 1H), 5.44 (s, 1H), 5.10 (t, J = 5.9 Hz, 1H), 4.95 (d, J = 4.8 Hz, 1H), 4.51 (dd, J = 19.5, 6.2 Hz, 1H), 4.37 (p, J = 7.0 Hz, 1H), 4.30 - 4.14 (m, 5H), 4.14 - 4.02 (m, 1H), 3.86 (s, 2H), 2.74 - 2.55 (m, 1H), 2.35 - 2.18 (m, 2H), 2.08 - 1.96 (m, 1H), 1.77 - 1.61 (m, 3H), 1.60 - 1.49 (m, 1H), 1.50 (s, 3H), 1.27 (d, J = 7.1 Hz, 3H), 1.23 (d, J = 7.1 Hz, 3H), 0.86 (s, 3H).

[0145] Step 2: (9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl Synthesis of 4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate.

[0146] Di-tert-butyl N,N-diethylphosphoramidite (1.8 mL, 6.02 mmol) was dissolved in 100 ml of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8 To a room temperature solution of a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (1.98 g, 2.041 mmol) and tetrazole (0.45 M in acetonitrile) (18 mL, 8.10 mmol) was added. After 50 min, the reaction was treated with hydrogen peroxide (30% in water, 1.2 mL, 11.75 mmol). After 10 min, the reaction was treated with 1 M aqueous NaSO (80 mL) to quench any unreacted hydrogen peroxide [Caution: Exothermic]. The mixture was extracted with EtOAc (3 x 60 mL). The combined organic layers were washed with brine (50 mL), dried (NaSO), filtered, and the solvent removed under reduced pressure. Purification by chromatography (silica, 120 g) eluting with a gradient of 0-10% MeOH / DCM afforded the desired product as a white foam (1.85 g, 78% yield). LCMS (Method d, ESI, Table 1) R t =1.12 min, m / z1162.7(M+H + ).

[0147] Step 3: Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl di-tert-butyl phosphate.

[0148] Piperidine (1.0 ml, 10.10 mmol) was dissolved in tetrahydrofuran (16 mL) to prepare (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a- To a room temperature solution of dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (1.854 g, 1.595 mmol) was added. After 25 minutes, the solvent was removed under reduced pressure. The residue was repeatedly treated with toluene (3 x 30 mL), which was removed under reduced pressure after each treatment to ensure complete removal of piperidine. The yield was assumed to be 100%, and the crude product was used in the next step without further purification. LCMS (Method d, ESI, Table 1) showed 100% purity. t =1.11 min, m / z940.7(M+H + ).

[0149] Step 4: Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl di-tert-butyl phosphate.

[0150] EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline) (1.630 g, 6.59 mmol) was added to a room temperature solution of bromoacetic acid (0.680 g, 4.90 mmol) in dimethylformamide (4 mL). The mixture was stirred for 90 min to activate the bromoacetic acid for coupling. Separately, 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl di-tert-butyl phosphate (1.499 g, 1.595 mmol) was dissolved in dimethylformamide (8 mL). This solution was transferred to the vial containing activated bromoacetic acid. Because the reaction was incomplete, a second feed of activated bromoacetic acid was prepared: bromoacetic acid (0.363 g, 2.61 mmol) was added to a solution of EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline) (0.813 g, 3.29 mmol) in dimethylformamide (3 mL), stirred for 70 minutes, and then combined with the reaction mixture. After 15 minutes, the reaction was diluted with EtOAc (60 mL), washed with water (2 × 20 mL), dried (NaSO), filtered, and the solvent removed under reduced pressure. Purification by chromatography (silica, 80 g) eluting with a gradient of 0–10% MeOH / DCM afforded the product as a white solid (0.760 g, 45% yield). LCMS (Method c, ESI, Table 1) IR t =1.03 min, m / z1060.6, 1062.1(M+H + ). 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.47 (d, J = 7.3 Hz, 1H), 8.22 (d, J = 7.2 Hz, 1H), 7.49 - 7.43 (m, 1H), 7.41 (t, J = 1.9 Hz, 1H), 7.35 (d, J = 8.2 Hz, 2H), 7.32 - 7.23 (m, 3H), 7.19 (t, J = 7.8 Hz, 1H), 6.91 (dt, J = 7.7, 1.4 Hz, 1H), 6.30 (dd, J = 10.2, 1.9 Hz, 1H), 6.12 (d, J = 2.2 Hz, 1H), 5.74 - 5.55 (m, 2H), 5.56 (s, 1H), 5.03 - 4.90 (m, 2H), 4.65 (dd, J = 18.0, 9.3 Hz, 1H), 4.34 (dt, J = 15.6, 7.2 Hz, 2H), 4.26 - 4.16 (m, 1H), 3.94 - 3.86 (m, 4H), 3.31 (s, 1H), 2.75 - 2.56 (m, 1H), 2.35 - 2.17 (m, 2H), 2.07 (d, J = 13.2 Hz, 1H), 1.72 (td, J = 13.9, 4.2 Hz, 2H), 1.67 - 1.52 (m, 1H), 1.49 (s, 3H), 1.42 (s, 9H), 1.42 (s, 9H), 1.27 (d, J = 7.1 Hz, 3H), 1.20 (d, J = 7.0 Hz, 3H), 0.89 (s, 3H).

[0151] Step 5: Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate.

[0152] Trifluoroacetic acid (2.5 mL, 32.4 mmol) was dissolved in dichloromethane (5 mL) to give 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl To a room temperature solution of di-tert-butyl phosphate (0.760 g, 0.716 mmol) was added. After stirring in an open vessel for 15 minutes, the solvent was removed under reduced pressure to give a foam, which was dissolved in 3:1 acetonitrile / water (16 mL) and purified by preparative HPLC on a Phenomenex C18(2) 10 micron column (250 x 50 mm). A gradient of acetonitrile (A) and 1% trifluoroacetic acid in water (B) was used at a flow rate of 90 mL / min (0-5.0 min, 15% A, 5.0-20.0 min, linear gradient 15-95% A, 5 min hold). The product fractions were freeze-dried to give the title compound as a white solid (0.291 g, 43% yield). LCMS (Method c, APCI, Table 1) R t =0.84 min, m / z948.4, 950.4(M+H + ). 1H NMR (600 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.46 (d, J = 7.4 Hz, 1H), 8.21 (d, J = 7.3 Hz, 1H), 7.46 (ddd, J = 8.2, 2.2, 1.1 Hz, 1H), 7.39 (t, J = 1.9 Hz, 1H), 7.38 - 7.33 (m, 2H), 7.29 - 7.23 (m, 3H), 7.19 (t, J = 7.8 Hz, 1H), 6.91 (dt, J = 7.7, 1.3 Hz, 1H), 6.30 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (dt, J = 2.6, 1.3 Hz, 1H), 5.64 (dddd, J = 48.3, 11.1, 6.4, 1.9 Hz, 2H), 5.53 (s, 1H), 4.96 - 4.89 (m, 2H), 4.59 (dd, J = 18.1, 8.4 Hz, 1H), 4.34 (dp, J = 23.2, 7.1 Hz, 2H), 4.21 (d, J = 9.0 Hz, 1H), 3.93 - 3.88 (m, 4H), 2.72 - 2.61 (m, 1H), 2.32 - 2.20 (m, 2H), 2.09 - 2.03 (m, 1H), 1.72 (dd, J = 12.8, 6.3 Hz, 2H), 1.68 (s, 1H), 1.57 - 1.49 (m, 1H), 1.49 (s, 3H), 1.27 (d, J = 7.1 Hz, 3H), 1.21 (d, J = 7.1 Hz, 3H), 0.88 (s, 3H).

[0153] 9.2.4. Conjugation and Analysis Methods

[0154] 9.2.4.1. Small Molecule Analysis Procedures High performance liquid chromatography (HPLC) and LCMS analytical data are detailed within the experiments or with reference to the conditions listed in Table 3.

[0155] [Table 3]

[0156] CD19 Phosphorylation GRM Agonist ADC 170.4 g of the CD19 antibody (CD19 mAb) of Example 1 at a concentration of about 50 mg / mL was diluted 1:1 w / w with PBE (125 mM phosphate, 6 mM EDTA, pH 7.3) buffer and partially reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (2.6 equivalents, 0.847 g, 25 mM solution in water for injection (WFI)) overnight at about 4° C. under nitrogen. The reduced antibody and a compound of Formula V:

[0157] [ka] Conjugation of the drug linker (6.0 equiv., 6.92 g, 93.2% w / w%, solution in 650 g of dimethylacetamide (DMA)) was carried out at approximately 4 °C by adding the drug linker, warming to 20 °C, and holding for 6-8 hours until deemed complete. The conjugation was quenched by adding N-acetylcysteine ​​(20 equiv., 3.71 g, 100 mM solution in WFI). The product solution was divided into two portions, each of which was then diluted to approximately 13 L with WFI and the pH adjusted to approximately 8.5 with NaOH to form the loading solution. Each loading solution was purified.

[0158] 9.2.4.3 Mass spectrometry The ADC samples were fully reduced before MS analysis. The mass spectrometry conditions used were as follows: HPLC column: Waters BioResolve RP mAb Polyphenyl 450A 2.7 μm, 150 × 2.1 mm; mobile phase A: 0.1% difluoroacetic acid in water; mobile phase B: 0.1% difluoroacetic acid in acetonitrile; flow rate: 0.4 mL / min; gradient: starting at 20% B, 20–45% B in 0–12 min, 45–60% B in 14–16 min, 60–100% B in 16–18 min, re-equilibrated to 20% B over 4 min; column temperature: 80 °C; MS ionization source: ESI.

[0159] Reduced reversed-phase (A280) chromatographs and deconvoluted mass spectra of each subunit were generated (see Figure 16). Peaks corresponding to light chains with zero or one drug linker molecule attached and heavy chains with zero, one, two, or three drug linker molecules attached were resolved and identified.

[0160] CD19-Dephosphorylating GRM Agonist ADCs 2.82 g of the CD19 antibody (CD19 mAb) of Example 1 at a concentration of about 50 mg / mL was diluted 1:1 w / w in PBE (125 mM phosphate, 6 mM EDTA, pH 7.3) buffer and partially reduced with TCEP (2.56 equivalents, 0.0141 g, 4.93 mM solution in WFI) overnight at about 4° C. under nitrogen. The reduced antibody and a compound of Formula VI were then reacted.

[0161] [ka] Conjugation of the drug linker (6.1 equiv., 0.102 g, 90.2% w / w%, solution in 11 g of DMA) was carried out at approximately 4 °C by adding the drug linker, followed by warming to 20 °C and holding for 6-8 hours until deemed complete. The conjugation was quenched by adding N-acetylcysteine ​​(10 equiv., 0.031 g, 100 mM solution in WFI). The product solution was salted with 0.8 M ammonium sulfate, 25 mM sodium phosphate, pH 7.1 (126 g) to prepare a loading solution for purification. The loading solution was purified.

[0162] 9.3 Example 3: Characterization of antibodies and ADCs The binding and intracellular activity of the CD19-phosphorylated GRM agonist ADC (ADC-1) and its parent defucosylated antibody (also referred to as CD19 mAb) described in Example 2, Section 8.2.4.2 were characterized. ADC-1 and CD19 mAb bind to CD19 endogenously expressed on the surface of multiple B-cell lymphoma cell lines. ADC-1 elicited highly potent antiproliferative effects and ADCC activity against multiple B-cell malignant cell lines. Furthermore, ADC-1 activated NFAT ADCC reporter cells expressing the F158 or V158 FcγRIIIa variant with similar potency. ADC-1 showed negligible binding to mouse or rat CD19, while showing high-affinity binding to human and cynomolgus monkey (cyno) CD19 and activating GRE reporter cell lines expressing human or cyno CD19.

[0163] 9.3.1 Materials and Methods

[0164] 9.3.1.1. Antibodies, Proteins, and Compounds A humanized anti-CD19 antibody (CD19 mAb) was defucosylated using ProBiogen technology. ADC-1 was produced by conjugating the CD19 mAb with a drug linker of formula V. GLP materials of CD19 mAb and ADC-1 were used in all experiments. Negative controls tested included the defucosylated anti-tetanus toxoid antibody AB095 (isotype mAb) and its ADC consisting of AB095 conjugated with a drug linker of formula V (isotype-GRM). Compounds analyzed included the GRM of formula IV, dexamethasone (Clinigen, cat#00641-0367-25), and prednisolone (Clinigen, cat#44523-0182-08).

[0165] 9.3.1.2.Cell culture The cancer cell lines Raji (Burkitt's lymphoma; ATCC, cat# CCL-86), SUP-B15 (acute lymphoblastic leukemia; ATCC, cat# CRL-1929), RS4;11 (acute lymphoblastic leukemia; ATCC, cat# CRL-1873), SU-DHL-6 (diffuse large B-cell lymphoma; ATCC, cat# CRL-2959), and HCT116 (colorectal carcinoma; ATCC, cat# CCL-247) were cultured in RPMI-1640 medium (Thermo Fisher Scientific, cat# A1049101) supplemented with 10% FBS (Thermo Fisher Scientific, cat# 26140079). The cancer cell line OCI-LY19 (diffuse large B-cell lymphoma; DSMZ, cat#ACC 528) was cultured in IMDM medium with 10% sterile-filtered human serum from platelet-poor plasma (Sigma-Aldrich, cat#P2918-100ML). 293T (ATCC, cat#CRL-3216) cells were cultured in DMEM supplemented with 10% FBS. All cells were cultured in a humidified chamber at 37°C and 5% CO2.

[0166] 9.3.1.3. Generation of Engineered Cell Lines The K562 GRE luciferase reporter cell line expressing human CD19 was generated at AbbVie. Briefly, K562 cells were seeded at 500,000 cells per well into a 6-well dish (Costar, cat#3516) with 2 mL of complete growth medium [RPMI 1640 supplemented with L-glutamine (Thermo Fisher Scientific, cat#11835-030), 10% FBS (Thermo Fisher Scientific Inc., cat#26140-079), 1% Na-pyruvate (Thermo Fisher Scientific Inc., cat#11360-070), and 1% MEM-NEAA (Thermo Fisher Scientific Inc., cat#111140-50)] and incubated at 37°C and 5% CO for 24 hours. The next day, 1.5 μg of pGL4.36[Luc2P / MMTV / Hygro] (Promega cat#E316) and 3 μL of PLUS Reagent (Thermo Fisher Scientific, cat#10964-021) were diluted in 244 μL of Opti-MEM (Thermo Fisher Scientific, cat#31985-070) and incubated at room temperature for 15 minutes. After incubation, the diluted DNA solution was preincubated with 13.2 μL of 1:1 Lipofectamine LTX solution (Thermo Fisher Scientific, cat#94756) (13.2 μL + 256.8 μL of Opti-MEM) and incubated at room temperature for 25 minutes to form the DNA-Lipofectamine LTX complex. After incubation, 500 μL of the DNA-Lipofectamine complex was added directly to the wells containing the cells. Transfection into K562 cells was performed for 24 hours at 37°C and 5% CO2. After incubation, cells were washed with 3 mL of PBS and placed under selection for 2 weeks in complete growth medium containing 125 μg / mL hygromycin B (Thermo Fisher Scientific, cat#10687-010).

[0167] K562 pGL4.36[Luc2P / MMTV / Hygro] cells were harvested and seeded at 250,000 cells per well in a 15 mL conical tube (Costar, cat#3516) with 1 mL of complete growth medium (RPMI+L-Glu, 10% FBS, 1% Na-pyruvate, and 1% MEM-NEAA). 3 μg of human CD19 (Origene, cat#RC230267) and 3 μL of PLUS Reagent (Thermo Fisher Scientific, cat#10964-021) were diluted in 244 μL of Opti-MEM (Thermo Fisher Scientific, cat#31985-070) and incubated at room temperature for 5 minutes. After incubation, the diluted DNA solution was preincubated with 11 μL of 1:1 Lipofectamine LTX solution (Thermo Fisher Scientific, cat#94756) (11 μL + 239 μL of Opti-MEM) and incubated at room temperature for 15 minutes to form the DNA-Lipofectamine LTX complex. Subsequently, 500 μL of the DNA-Lipofectamine complex was added directly to the conical tube containing the cells. The cells and DNA-Lipofectamine were mixed, seeded into 6-well dishes (Costar, cat#3516), and incubated at 37°C and 5% CO for 24 hours. After 24 hours of incubation, cells were selected for 2 weeks using 2 ml of complete growth medium containing 125 μg / mL hygromycin B (Thermo Fisher Scientific, cat#10687-010) and 225 μg / mL G418 (Thermo Fisher Scientific, cat#10131-027).

[0168] 9.3.1.4. Cell proliferation assay Adherent cells (293T and HCT116) were detached by trypsinization. 1,000–2,000 cells were plated per well in 40 μL of complete RPMI 1640 in 384-well tissue culture plates (Corning, cat#3764). Adherent cells were allowed to attach overnight. The following day, cells were treated with the indicated concentrations of drugs in 10 μL and incubated at 37°C, 5% CO2 for 5 days. At the endpoint, 40 μL of CellTiter-Glo Luminescence Cell Viability Assay Reagent (Promega, cat#G7573) was added per well. Plates were read on a Molecular Devices SpectraMax M5 plate reader using the Luminescence setting. Cell viability was expressed as a percentage of the untreated control.

[0169] Immunofluorescence The RS4;11 cell line was plated at 50,000 cells per well in V-bottom plates (Costar Cat#3894). Cells were Fc-blocked using PBS + 5% normal human serum (Sigma Cat#H4522) and 2% FBS. Cells were washed and resuspended in staining buffer (PBS + 2% FBS). Cells were stained with 5 μg / mL of anti-hu-CD19 antibody (CD19 mAb) conjugated with a dye (Alexa Fluo 647 dye) in staining buffer and incubated on ice for 1 hour. After incubation, cells were washed three times with PBS containing 2% FBS and chased at 37°C for 0.5, 1, 3, and 24 hours to induce internalization and compared to cells stained on ice as a T=0 time point control. Cells were then fixed and permeabilized using the manufacturer's instructions (BD, Cat#554714). The cells were blocked with staining buffer containing 1% saponin and 2% mouse serum (Invitrogen Cat#3881) and stained overnight at 4°C with 2 μg / mL of LAMP1 antibody (Biolegend, Cat#328610) conjugated with a fluorescent dye (Alexa Fluor 488 dye). The cells were washed three times in PBS containing 2% FBS. The cells were resuspended in imaging medium (FluroBrite: Gibco Cat#A1896701), transferred to a glass-bottom imaging plate (Thermo Fisher Scientific, Cat#160376), and analyzed using a confocal imager (IMX Micro Confocal Imager).

[0170] Immunoblotting SU-DHL-6 cells were treated with isotype mAb or CD19 mAb for 1 hour and then stimulated with 1 μg / ml anti-IgM for 0, 5, 15, or 30 minutes. Cells were harvested by washing twice with ice-cold PBS and then lysed in RIPA lysis buffer (Sigma-Aldrich, cat#R0278) supplemented with 1x Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, cat#78446). Protein concentrations of the lysates were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, cat#23227). 10-20 μg of lysate was separated on a 4-12% gradient gel (Thermo Fisher Scientific, cat#NW04120BOX) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, cat#IB23001). The membrane was incubated overnight at 4°C with primary antibodies at the concentrations recommended by the manufacturer. Primary antibodies used in this study included phospho-AKT Ser473 (Cell Signaling Technology, cat#4060) or GAPDH (Cell Signaling Technology, cat#97166). The membrane was washed three times with 1x PBST (Cell Signaling Technology, cat#9809) and then incubated for 1 hour at room temperature with secondary antibodies (Peroxidase AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, cat#115-035-003) or Peroxidase AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, cat#111-035-003)). The membrane was washed three times with 1x PBST and then incubated with Western Blot Substrate (Pierce™ ECL Western Blotting Substrate, Thermo Fisher Scientific, cat#32106).The membrane was then subjected to detection using a Western blot imaging system (Azure Image Systems C600).

[0171] 9.3.1.7. Glucocorticoid Response Element (GRE) Activation Assay K562 parental GRE cells (pGL4.36[luc2P / MMTV / Hygro]) and K562 human CD19 GRE cells (pGL4.36[luc2P / MMTV / Hygro]) or K562 cynomolgus monkey CD19 GRE cells (pGL4.36[luc2P / MMTV / Hygro]) were plated at 50,000 cells per well in 75 μL of assay medium (1x RPMI + L-Glu, 1% CSFBS (Thermo Fisher Scientific Inc., cat#12676-029), 1% Na-pyruvate, and 1% MEAA) in 96-well tissue-culture-treated white plates (Costar, cat#3917). GRE K562 parental cells or cells expressing human CD19 and cells expressing cynomolgus monkey CD19 were treated with 25 μL of CD19-GRM serially diluted 4-fold in assay medium and incubated for 72 hours at 37°C and 5% CO2. 100 nM dexamethasone was added to these assays as a positive control. After 72 hours of incubation, cells were treated with 100 μL of the Dual-Glo Luciferase Assay System (Promega cat#E2920) for 10 minutes and analyzed for luminescence using a MicroBeta (PerkinElmer). Dose-response data were fitted to a sigmoidal curve using nonlinear regression to determine the EC 50 Values ​​were calculated using GraphPad 9.0 (GraphPad Software, Inc.).

[0172] NFAT ADCC Reporter Assay ADCC reporter bioassays (V and F variants; Promega, cat#G7010 and G9790) were performed according to the manufacturer's protocol with minor modifications. Briefly, the target cell line Raji was plated at 2500 cells per well in 5 μL of ADCC assay buffer (RPMI medium containing 4% low IgG serum). Antibodies were titrated in ADCC assay buffer, and 5 μL was added at the indicated antibody concentrations. Effector cells from the V variant kit and F variant kit were resuspended in ADCC assay buffer and added at 5 μL per well to achieve an effector-to-target cell ratio of 6:1. The target cell-antibody mixture was incubated with effector cells expressing the V variant and effector cells expressing the F variant at 37°C for 6 and 24 hours, respectively. At the endpoint, 15 μL of Bio-Glo Luciferase Reagent was added and luciferase activity was measured using a Molecular Devices SpectraMax M5 plate reader.

[0173] 9.3.1.9. PBMC Co-culture ADCC Assay Target cell lines (RS4;11, Raji, and KARPAS422) were washed with PBS and labeled with 1 μM CFSE (Thermo Fisher Scientific, cat#C34554) in PBS for 5 minutes at 37°C. The labeled target cells were then washed three times with medium containing 10% FBS. 20,000 labeled target cells in 50 μL were plated in a V-shaped 96-well plate. Antibodies and ADCs were titrated in culture medium, and 10 μL was added to achieve the indicated final concentrations. PMBCs from a normal donor (AllCells) were added in 40 μL at an effector-to-target cell ratio of 20:1, and the cells were incubated at 37°C for 4 hours. After incubation, cells were washed once with PBS and stained with the Live / Dead Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific cat#L34955) for 30 minutes at room temperature, then washed once with 200 μL of ice-cold PBS. Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes and then washed once with 200 μL of PBS. Fixed cells were resuspended in 100 μL of PBS and stored at 4°C until analysis on a Stratedigm S1000EON flow cytometer. The percentage of Live / Dead Violet dye-positive cells was captured, and % specific lysis was calculated by subtracting the percentage of Live / Dead Violet dye-positive cells in each treatment condition from the untreated control containing only effector and target cells.

[0174] ADCP Assay Human monocytes (STEMCELL Technologies) were differentiated into macrophages for 8 days in RPMI-1640 containing 10% FBS and 50 ng / ml recombinant human M-CSF (BioLegend). Fully differentiated macrophages were detached from the flask and co-cultured with target cancer cells in a phagocytosis assay. Human non-Hodgkin's lymphoma cell lines Raji and NuDHL1, which express endogenous CD19, were selected as target cells. After washing with PBS, the target cancer cells were labeled with 5 μM CFSE dye solution (Thermo Fisher Scientific) for 15 minutes and then co-incubated with human macrophages at a 1:1 ratio. CD19 mAb or CD19-GRM was added at various concentrations. After 3 hours of coculture at 37°C, cells were harvested, washed, and stained with an antibody against macrophage maturation marker, APC-Cy7-labeled CD68 antibody (BioLegend), followed by flow cytometry analysis. The phagocytosis index, which indicates the population percentile of phagocytic macrophages, was calculated by counting the number of CFSE+CD68+ double-positive macrophages among total CD68+ macrophages.

[0175] 9.3.1.11. Apoptosis Assay Apoptosis was detected using Western blot assays. DLBCL cell lines, Farage, SU-DHL-6, and OCI-LY19, were treated with GRM payload (100 nM) or ADC-1 (1 μM) for the indicated times, followed by cell lysis. Cell lysates were generated using protein extraction reagent (M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, cat#78501)), and protein concentrations were determined using a Bradford assay (Pierce™ Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific, cat#23246)). Protein lysates (10 μg) from each treatment condition were separated on a polyacrylamide gel (Bolt™ 4-12%, Bis-Tris) and transferred to a PVDF membrane using an iBlot™ 2 Gel Transfer Device. The membranes were blocked with 5% BSA and then immunoblotted with antibodies against the proteins of interest: BIM (Cell Signaling, cat#2933), caspase 3 (Cell Signaling, cat#9662), PARP (Cell Signaling, cat#9532), and GAPDH (Cell Signaling, cat#5174). The membranes were then probed with horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit or mouse IgG and detected using a chemiluminescent horseradish peroxidase substrate (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, cat#34580) on an Azure Imager Western blot imaging system.

[0176] 9.3.2. In vivo testing 9.3.2.1. Mouse and Management Female CB17 / SCID and SCID / beige mice were obtained from Charles River (Wilmington, MA), and CD34 + Humanized NSG IL-15 mice were obtained from the Jackson Laboratory (Bar Harbor, ME) at 6-8 weeks of age and housed in cages of up to 10 mice. They weighed 18-20 g upon arrival. Food and water were available ad libitum. Mice were acclimated to the animal facility for at least 3 days prior to the start of experiments. Animals were tested under a 12-hour light:12-hour dark schedule (lights on at 06:00 hours) during the photoperiod. All experiments were performed in accordance with AbbVie's Institutional Animal Care and Use Committee and the National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines.

[0177] 9.3.2.2. Compounds The intraperitoneal route of administration was used. For GRM small molecules (GRM-SM), formulated in 0.05% HPMC, 0.02% Tween-80 in water and stored at 4° C., oral dosing was used.

[0178] The CD19 mAb of Example 1 is a defucosylated monoclonal antibody (IgG1, κ) with high affinity for human and cynomolgus monkey CD19. ADC-1 is a conjugate of CD19 mAb linked to a GRM molecule of Formula V using a bromoacetamide alanine-alanine peptide linker. The isotype antibody (AB095) recognizes a tetanus toxoid antigen that is not presented by xenografts or any mouse tissue.

[0179] 9.3.2.3. Efficacy Parameters To refer to the efficacy of a drug, a parameter of the magnitude of the therapeutic response (maximum tumor growth inhibition [TGI max]) and long-term parameters (tumor growth delay, TGD) were used. TGI represents the difference between the mean tumor volume of a drug-treated group and the mean tumor volume of a control group treated with drug vehicle or isotype-matched non-binding antibody, and is expressed as a percentage of the mean volume of the control group. max The value is determined at the point where the difference between the treatment and control groups is greatest. (n) is the defined tumor volume (1 cm) in the drug treatment group. 3 ) compared to the median time to reach the same volume in a vehicle-treated control group. The difference is expressed as a percentage of the median time to reach the specified tumor volume in the control group.

[0180] 9.3.2.4. Generation of Tumor-Bearing Mice with Subcutaneous Flank Tumors and Determination of Tumor Volume For each subcutaneous xenograft study, mice were inoculated with viable cells into the right flank on day 0. The injection volume was 0.1 mL and consisted of a 1:1 mixture of S-MEM or HBSS (Fisher Scientific, MA) and Matrigel (BD, Franklin Lakes, NJ). Tumor size was approximately 80–200 mm. 3 Treatment was usually initiated within 24 hours after randomization into the required cohorts and tumor size balancing. Mice weighed approximately 20-22 g at the start of treatment. Tumor volume was estimated once or twice weekly. Tumor length (L) and width (W) measurements were obtained with an electronic caliper, and volume was calculated according to the following equation: V = (L × W) 2 ) / 2. Tumor volume up to 2,000mm 3 Mice were euthanized when they reached 0.05 mg / kg or if their health was compromised according to institutional guidelines.

[0181] Statistics Data from in vivo experiments were analyzed using the TGI maxValues ​​were analyzed using Student's t-test and TGD using the log-rank test (Mann-Whitney U test or Mantel-Cox). Differences in response rates were assessed by Fisher's exact test.

[0182] PDX studies Patient-derived xenograft (PDX) studies were performed on DLBCL models established at WuXi, originating from surgically resected primary patient tumors. Patient treatment histories were collected in accordance with protocols and guidelines established by the Institutional Review Board. Each tumor specimen (single-cell suspension or tumor fragments) was prepared in Matrigel (BD Biosciences) and implanted into the flank of 6-8 week-old NOD scid mice. Mice were handled and housed in accordance with IACUC / AALAS protocols and procedures. Tumors were measured twice weekly by caliper measurements (length × width). 2 ) / 2, and tumor volume was approximately 150-200 mm 3 Treatment was initiated when the tumor volume reached 100%. Mice were treated intraperitoneally once with the indicated test article (vehicle, CD19 antibody, ADC-1) at n=5 / group. For each treated DLBCL PDX model (six germinal center B cell (GCB) and four non-GCB, including the difficult-to-treat non-GCB activated B cell subtype (ABC)), tumor growth inhibition after treatment with ADC-1 was determined as follows: delta %TGI max = 1 - (treatment group tumor volume on day X - treatment group tumor volume at randomization) / (control tumor volume on day X - control tumor volume at randomization)] * 100. Delta %TGI max was determined when the difference between the treatment and control groups was greatest. PDX models were determined as responders or non-responders based on their ability to induce tumor regression (responder): >105% delta TGI max - responder / no responder. The percent change in tumor volume was also calculated as [(tumor volume at time point / tumor volume at randomization)−1]×100, with vehicle control tumors at 1000 mm 3 The graphs are based on the time point at which the blood pressure reached 100°C. See Figures 13A-B.

[0183] 9.3.2.7. Binding activity Flow cytometry was used to assess cell surface binding of ADC-1 to different cancer cell lines endogenously expressing CD19 and to 293T cells transduced with full-length human CD19. ADC-1 showed binding EC values ​​ranging from 0.544 to 1.061 nM to the B-cell lymphoma cell lines Raji (Burkitt's lymphoma), SUP-B15 (acute lymphoblastic leukemia), RS4;11 (acute lymphoblastic leukemia), and OCI-LY19 (diffuse large B-cell lymphoma). 50 (Table 4) and showed a binding EC of 1.086 nM against 293T cells engineered with full-length human CD19. 50 The unconjugated parent defucosylated CD19 antibody (CD19 mAb) of Example 1 exhibited an EC range of 0.401 to 0.836 nM (Table 4). 50 (Table 4) and a binding EC50 of 0.977 nM for 293T cells engineered with full-length human CD19. 50 Neither ADC-1 nor CD19 mAb showed detectable binding to the CD19-negative colorectal cancer cell line HCT116 (Table 4).

[0184] [Table 4]

[0185] 9.3.2.8. Glucocorticoid Receptor Modulator (GRM) Payloads Are More Potent Than Clinically Approved Glucocorticoids The efficacy of the GRM payload was compared to clinically approved glucocorticoids (dexamethasone and prednisolone) by testing in vitro on RS4;11 cells. The GRM payload of Formula IV demonstrated enhanced efficacy compared to either dexamethasone or prednisolone (Figure 1). EC 50 were 0.23 nM, 3.28 nM, and 82.91 nM for the GRM payloads of Formula IV, dexamethasone, and prednisolone, respectively.

[0186] 9.3.2.9. CD19 mAb and ADC-1 Internalize, Deliver GRM Payloads, and Inhibit B-Cell Lymphoma Cell Growth To investigate the ability of CD19 mAbs to induce CD19 internalization, the acute lymphoblastic lymphoma (ALL) cell line RS4;11 was treated with CD19 mAbs for 0, 0.5, 1, 3, and 24 h at 37°C. As indicated by colocalization of CD19 with the lysosomal marker LAMP1, CD19 mAbs induced CD19 internalization and localization to lysosomes as early as 0.5 h (Figure 2). Similarly, ADC-1 dose-dependently activated the glucocorticoid response element (GRE) reporter in K562 GRE reporter cells engineered to express human CD19 (Figure 3).

[0187] To evaluate the cytotoxicity of ADC-1, proliferation assays were performed on cell lines expressing different levels of CD19. ADC-1 inhibited the proliferation of CD19-expressing cancer cell lines that were sensitive to the glucocorticoid receptor modulator (GRM) payload of Formula IV, and the in vitro cytotoxic EC of ADC-1 was significantly increased. 50 ranged from 0.082 to 0.321 nM (Table 5).

[0188] [Table 5]

[0189] The GRM prodrug payload of formula III was dephosphorylated in vitro / in vivo to give the dephosphorylated GRM payload of formula IV, and thus the dephosphorylated payload of formula IV was used as a control in our studies. ADC-1 was not active against the CD19-negative colorectal cancer cell line HCT116 (Table 5). Notably, ADC-1 was more potent than isotype-GRM (Table 5), indicating CD19 target-dependent delivery of the GRM payload.

[0190] 9.3.2.10. CD19 mAb inhibits DLBCL cell proliferation and AKT activation in vitro CD19 is a coreceptor for the B cell receptor and can transactivate the PI3K / AKT pathway (Burger et al., Nat Rev Cancer, 2018.18(3):148-167). Furthermore, functional genomic screening demonstrated that a subset of DLBCL cell lines is dependent on CD19 for survival (Phelan, JD et al., Nature, 2018.560(7718):387-391). In vitro, treatment with CD19 mAb (Example 1) inhibited SU-DHL-6 cell proliferation (Figure 4A). Pretreatment with CD19 mAb eliminated AKT phosphorylation in response to BCR stimulation with 1 μg / ml anti-IgM (Figure 4B), demonstrating that CD19 mAb effectively blocks BCR-mediated AKT pathway activation.

[0191] 9.3.2.11. ADC-1 Mediates Antibody-Dependent Cellular Cytotoxicity (ADCC) CD19 mAb and ADC-1 were defucosylated to enhance their ability to elicit antibody-dependent cellular cytotoxicity (ADCC). Polymorphisms in FcγRIIIa (F158 or V158) affect their binding to IgG1, IgG3, and IgG4, as well as the therapeutic efficacy of these antibodies (Koene, H.R. et al., Blood, 1997. 90(3):1109-1114; Cartron, G. et al., Blood, 2002. 99(3):754-8; Wu, J. et al., J Clin Invest, 1997. 100(5):1059-70).

[0192] In vitro, we determined the ability of CD19-GRM to bind to FcγRIIIa and elicit ADCC using NFAT reporter Jurkat cells engineered to express high affinity (V158) or low affinity (F158) FcγRIIIa. CD19-GRM induced ADCC NFAT reporter activation and EC 50The EC values ​​were 0.017 nM and 0.018 nM for the V158 and F158 variants, respectively (Figures 5A and 5B). Similarly, the unconjugated defucosylated CD19 mAb also induced highly potent ADCC activity, with EC 50 were 0.027 nM and 0.023 nM for the V158 and F158 variants, respectively (Fig. 5A and B).

[0193] The ADCC activity of ADC-1 was functionally assessed by co-culture of B-cell lymphoma cell lines with primary PBMCs. In these assays, ADC-1 elicited CD19-specific cancer cell lysis and inhibited EC 50 The EC values ​​ranged from 0.07 to 0.142 nM (Figures 6A-C). Unconjugated CD19 mAbs also showed similar CD19-specific cancer cell lysis, with EC 50 ranged from 0.075 to 0.405 nM (Figure 6A-C).

[0194] 9.3.2.12. ADC-1 induces antibody-dependent cellular phagocytosis (ADCP) The ability of ADC-1 to induce ADCP was assessed by coculturing B-cell lymphoma cells with monocyte-derived macrophages in vitro. Treatment with both ADC-1 and CD19 mAb resulted in significantly greater ADCP induction in NuDHL1 and Raji than treatment with isotype mAb or isotype GRM (Fig. 7A and B).

[0195] 9.3.2.13. GRM and ADC-1 induce apoptosis in DLBCL cell lines In all three cell lines tested, GRM and ADC1 induced apoptosis, as measured by upregulation of pro-apoptotic protein (BIM) and induction of markers of apoptosis (cleaved caspase-3 and cleaved PARP) (see Figure 15).

[0196] 9.3.2.14. ADC-1 Elicits GRM-Driven Antitumor Activity and ADCC In Vivo ADC-1 is effective against tumors with cell surface expression of CD19. When administered as a single agent, ADC-1 inhibits the growth of subcutaneous xenografts of human tumor cell lines derived from B-cell malignancies, such as diffuse large B-cell lymphoma (DLBCL: OCI-LY19 and SU-DHL-6) and acute lymphoblastic leukemia (ALL: RS4;11) (Figures 8 and 9). Human xenografts were tested using immunocompromised CB17 / SCID mice (OCI-LY19, RS4;11: Figures 8A and 9), immunocompromised SCID / beige mice (SU-DHL-6: Figure 8B), and a humanized CD34+NSG-huIL-15 mouse model (OCI-LY19: Figure 10). ADC-1 is administered intraperitoneally as a single bolus and is effective in a dose-dependent manner. In DLBCL xenografts (Figure 8), all doses of ADC-1 and the glucocorticoid receptor modulator small molecule of Formula IV (GRM-SM) induced statistically significant (p<0.05) tumor growth inhibition greater than vehicle, isotype monoclonal antibody, human CD19 monoclonal antibody, and systemic steroid (prednisolone, daily dosing). Significant and durable responses were observed at the highest doses in DLBCL xenografts. AB095-GRM (isotype-GRM control) showed some growth inhibition at the two highest doses, which was significantly less than ADC-1 at the same doses (p<0.05). ADC-1 administered to an ALL xenograft model (Figure 9) demonstrated high sensitivity, with durable responses and statistically significant (p<0.05) tumor growth inhibition at lower doses. Tumor regression at dose 2 lasted for >100 days. All doses were well tolerated and no weight loss was observed.

[0197] ADC-1 is effective in a dose-dependent manner in an antibody-dependent cell cytotoxicity (ADCC)-competent mouse model (huCD34+NSG-huIL-15). In this mouse model, immunocompromised NSG mice were genetically engineered to express human IL-15 and engrafted with human PBMCs (CD34+). Human IL-15 functions to support NK cell viability and enable ADCC testing. In this mouse system, ADC-1 induced inhibition of the growth of subcutaneous xenografts of a human tumor cell line derived from DLBCL (OCI-LY19; Figure 10). Statistically significant (p<0.05) tumor growth inhibition and durable responses (>50 days) were observed at dose 3. The effects of ADC-1 on human peripheral blood B cells and NK cells were measured at baseline (4 days before dosing) and 1, 6, 13, 20, and 26 days after dosing. Both the number of human B cells per microliter of mouse blood and their percentage of total human CD45+ cells were reduced at all ADC-1 dose levels. The duration of B cell suppression was positively related to dose level. NK cell numbers were not significantly affected by ADC-1 at any dose level.

[0198] The efficacy of ADC-1 was also directly compared with the combination of CD19 mAb and systemic prednisolone in the RS4;11 xenograft model. In this model, high doses of CD19 mAb or prednisolone ([QD×5]×3) as single agents exhibited modest antitumor activity, whereas combining both agents resulted in more durable tumor growth inhibition (Figure 11). Notably, a single low-dose of ADC-1 induced long-lasting antitumor activity compared with the combination of CD19 mAb and systemic prednisolone ([QD×5]×3).

[0199] ADC-1 demonstrates antitumor efficacy in several different B-cell malignancy models: DB (FIG. 12A), SUPB15 (FIG. 12B), and RS4;11 (FIG. 12C). The antitumor efficacy of ADC-1 was directly compared to prednisolone, [QD×5]×3, or isotype-GRM ADC in DB and SUPB15 xenograft models grown in immunocompromised mice as indicated. Durable antitumor efficacy was observed in both DB and SUPB15 xenograft models, which was superior to the systemic steroid prednisolone and to the isotype-GRM ADC, confirming the CD19-targeting activity of ADC-1. The ALL tumor model, SUPB15 (FIG. 12B), demonstrates sustained antitumor activity (complete response) lasting for more than 50 days after a single ADC-1 dose. RS4;11 tumors (FIG. 12C) were grown to large sizes (>600 mm 3 ) and then treated with a single IP dose of ADC-1. This single ADC-1 administration resulted in complete tumor regression of these large tumors, which persisted for >40 days.

[0200] ADC-1 elicits antitumor activity in DLBCL PDX models As shown in Figure 13A, all 10 DLBCL PDX models demonstrated tumor growth inhibition compared to the same PDX models treated with a control. Nine of the 10 models demonstrated tumor regression using a single dose of ADC-1 at dose 3 (see Figure 13B). Antitumor efficacy was observed in both GCB and non-GCB patient-derived PDX models. Only one model, 0232, demonstrated no plateau or regression when treated with ADC-1 at dose 3. This PDX model was derived from a patient who received seven cycles of R-CHOP treatment. Three PDX models, 0367, 0016, and 0207, derived from R-CHOP relapsed patients who received four cycles of treatment, responded.

[0201] As shown in Figures 14A and 14B, a single dose of ADC-1 at dose 3 demonstrated antitumor activity compared to vehicle control and treatment with a CD19 antibody. In Figure 14A, the PDX model was derived from a patient with DLBCL, GCB, who received four cycles of R-CHOP treatment. This PDX model showed little equilibrium with treatment with ibrutinib. The PDX-derived model shown in Figure 14B was derived from a patient with DLBCL, ABC, who received four cycles of R-CHOP. This PDX model was resistant to ibrutinib treatment.

Claims

1. an anti-CD19 antibody comprising a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and a light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain; the CDR-H1 domain comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1); the CDR-H2 domain comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2); the CDR-H3 domain comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3); the CDR-L1 domain comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4); the CDR-L2 domain comprises the amino acid sequence WASTRHT (SEQ ID NO:5); An anti-CD19 antibody, wherein the CDR-L3 domain comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6).

2. The anti-CD19 antibody of claim 1 , wherein the antibody is an IgG1 antibody.

3. 2. The anti-CD19 antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO:

8.

4. The anti-CD19 antibody of claim 3, wherein the antibody is an IgG1 antibody.

5. The antibody two heavy chains, each consisting of the amino acid sequence set forth as SEQ ID NO:9; and two light chains, each consisting of the amino acid sequence set forth in SEQ ID NO: 10; The anti-CD19 antibody of claim 1, comprising:

6. The anti-CD19 antibody of claim 5, wherein the antibody is afucosylated.

7. 6. The anti-CD19 antibody of claim 5, wherein the antibody is afucosylated at position 303 of SEQ ID NO:

9.

8. The antibody two heavy chains, both heavy chains consisting of the amino acid sequence set forth as SEQ ID NO:11; and two light chains, each consisting of the amino acid sequence set forth in SEQ ID NO: 10; The anti-CD19 antibody of claim 1, comprising:

9. The anti-CD19 antibody of claim 8, wherein the antibody is afucosylated.

10. 9. The anti-CD19 antibody of claim 8, wherein the antibody is afucosylated at position 303 of SEQ ID NO:

11.

11. The antibody two heavy chains, one heavy chain consisting of the amino acid sequence set forth as SEQ ID NO:9 and the other heavy chain consisting of the amino acid sequence set forth as SEQ ID NO:11; and two light chains, each consisting of the amino acid sequence set forth in SEQ ID NO: 10; The anti-CD19 antibody of claim 1, comprising:

12. The anti-CD19 antibody of claim 11, wherein the antibody is afucosylated.

13. 12. The anti-CD19 antibody of claim 11, wherein the antibody is afucosylated at position 303 of SEQ ID NO:9 and position 303 of SEQ ID NO:11.

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