Anti-CD19 antibody drug conjugates

Abstract

The present disclosure provides anti-CD19 antibody drug conjugates (ADCs), compositions and methods including the use of such ADCs.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 366,520 filed on June 16, 2022 and U.S. Provisional Patent Application No. 63 / 488,302 filed on March 3, 2023, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml copy was created on March 3, 2023, is named 350794.46710 and is 20,903 bytes in size. 3. Technical field

[0005] The present application particularly relates to novel anti-CD19 antibody drug conjugates (ADCs) and methods for preparing the same. 4. Background Technology

[0006] CD19 is a B cell marker and a co-activator of the B cell receptor (BCR) complex, which activates downstream proteins such as PI3K, thereby producing a proliferative phenotype. Due to this mechanism, CD19 is a required B cell antigen, and CRISPR or RNAi loss of CD19 expression causes normal B cells and B cell cancer cell lines to be unable to survive. CD19 is a clinically validated target with high expression in B cell malignancies (e.g., DLBCL, FL, CLL).

[0007] Systemic glucocorticoids are used to treat B-cell malignancies. These systemic steroids have robust monotherapy activity at high doses, but can lead to chronic steroid-related toxicity, limiting dose and efficacy potential. Glucocorticoids act by binding to intracellular glucocorticoid receptors (GRs), which then transport to the nucleus where they bind to glucocorticoid response elements (GREs) that drive transcriptional activation. Steroid-induced transcriptional changes include upregulation of pro-apoptotic proteins (e.g., BIM, BAX), reduction of anti-apoptotic proteins (e.g., BCL2), and inhibition of NFKB proliferation signals, ultimately leading to apoptotic cancer cell death.

[0008] Therefore, 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). 5. Summary of the invention

[0009] The present invention provides antibody drug conjugates targeting B cells via anti-CD19 antibodies linked to a glucocorticoid receptor modulator (GRM) agonist payload, and provides novel methods 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 have the following different mechanisms of action: (1) targeted delivery of a 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 afucosylation. Unlike traditional cytotoxic ADCs, GRM payloads are agonists that bind to intracellular glucocorticoid receptors (GR), which are then transported to the nucleus to drive transcriptional events associated with sustained glucocorticoid response elements (GREs), including regulation of apoptosis regulators, resulting in payload-driven apoptotic cell death. ADCC is a very effective mechanism in B cell malignancies. In one embodiment, the anti-CD19 antibody of ADC is engineered to maintain increased ADCC activity after being conjugated to the GRM agonist payload. By engineering the molecule in this way, anti-CD19 GRM agonist ADCs are multimodal antibody drug conjugates designed to have robust single agent activity and provide significant clinical benefits in a variety of B cell malignancies.

[0010] In certain aspects, the present invention provides an anti-CD19 antibody-drug conjugate comprising the following structure:

[0011]

[0012] in:

[0013] A is antibody;

[0014] R is the point of attachment of the antibody formed through a cysteine ​​residue of the antibody, which upon attachment provides an -S- group;

[0015] AA1 and AA2 are alanine (Ala);

[0016] R 1 is hydrogen or -P(=O)(OH)2;

[0017] n is an integer from 1 to 10;

[0018] Wherein the antibody is an anti-CD19 antibody, which comprises

[0019] a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and

[0020] A light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein

[0021] CDR-H1 comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1);

[0022] CDR-H2 comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2), and CDR-H3 comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3);

[0023] CDR-L1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4),

[0024] CDR-L2 comprises the amino acid sequence WASTRHT (SEQ ID NO: 5), and

[0025] CDR-L3 comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6). 6. Description of the drawings

[0026] 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.

[0027] Figure 1 : In vitro cytotoxicity assay of GRM payloads compared to approved small molecule steroids (dexamethasone and prednisolone). Cell viability % of RS4;11 after 5 days of compound treatment relative to vehicle control is shown. EC values ​​for each compound are summarized in the table. 50 .

[0028] Figure 2 : CD19 mAb induces CD19 internalization into lysosomes. Immunofluorescence staining showed that CD19 receptor localized to the lysosomal compartment after CD19 mAb treatment in RS4;11. LAMP-1 was used as a marker for the lysosomal compartment.

[0029] Figure 3 ADC-1 induces transcriptional activation of the glucocorticoid response element (GRE) luciferase reporter gene. K562 cells expressing human CD19 were treated with ADC-1 for 72 hours. The maximum GRE activation % was calculated by dividing the GRE luminescence signal at each concentration by the GRE luminescence signal at 333 nM.

[0030] Figure 4: CD19 mAb inhibits DLBCL cell proliferation and AKT activation in vitro. A, Viability of SU-DHL-6 relative to vehicle control after 5 days of treatment with isotype or CD19 mAb. B, SU-DHL-6 cells were treated with isotype or CD19 mAb for one hour and then stimulated with 1 μg / ml anti-IgM for the indicated time before cell lysis. S473 phosphorylation on AKT was detected via immunoblotting, and GAPDH was used as a loading control.

[0031] Figure 5: CD19 mAb and ADC-1 induce NFAT activation in Jurkat reporter cells expressing FcγRIIIa V158 and F158 allotypes. A and B, Raji cells were treated with dose-titrated antibodies or ADCs and co-incubated with Jurkat cells expressing V158 (A) and F158 (B) FcγRIIIa variants. Luminescent signals after 4 hours (A) or 16 hours (B) of incubation are shown.

[0032] Figure 6: CD19 mAb and ADC-1 induce ADCC against B cell malignancy cell lines in an ex vivo PMBC co-culture ADCC assay. AC, RS4;11 (A), Raji (B), and KARPAS422 (C) were incubated with dose titrated antibodies or ADCs and co-incubated with primary PBMCs at an effector to target ratio (E:T) of 20:1 for 4 hours. Specific lysis of cells was calculated relative to untreated samples.

[0033] Figure 7: ADC-1 induces antibody-dependent cellular phagocytosis (ADCP) of B cell malignancy cell lines in co-culture with monocyte-derived macrophages. A and B, CFSE-labeled NuDHL1 (A) and Raji (B) cells were treated with 200 nM of the indicated agents and co-cultured with monocyte-derived macrophages for 3 hours. CD68+ was used as a marker for mature macrophages, and the phagocytic index represents the percentage of engulfed target cells (CFSE+CD68+) in the total mature macrophage (CD68+) gate.

[0034] Figure 8: Growth inhibition of xenografted diffuse large B-cell lymphoma (OCI-LY19 and SU-DHL-6) in immunocompromised mice by different 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. The volume of subcutaneous tumors was plotted against time after randomization and administration. ADC-1, isotype-GRM, huCD19 antibody, isotype antibody were administered as a single dose (QDx1) at different dose levels (dose 1-5, dose 2 is 3 times the dose of dose 1, dose 3 is 10 times the dose of dose 1, dose 4 is 30 times the dose of dose 1, and dose 5 is 50 times the dose of dose 1), in mice with OCI-LY19 subcutaneous tumors (average tumor volume 117-205mm 3 Growth inhibition was studied in mice bearing SU-DHL-6 tumors (A) and SCID beige mice (B). GRM-SM was administered as multiple daily doses (QDx5)3. Each point on the curve represents the average of seven (SU-DHL-6) or eight (OCI-LY19) tumors. Error bars depict the standard error of the mean.

[0035] Fig. 9 : Growth inhibition of xenografted acute lymphoblastic leukemia (RS4;11) by different doses of ADC-1. Growth inhibition of xenografted RS4;11 tumors (immunocompromised CB17 / SCID mice) by administration of ADC-1. The volume of subcutaneous tumors was plotted against time after randomization and dosing. In subcutaneous tumors with xenografted RS4;11 cells (mean tumor volume 118 mm 3 ) mice, growth inhibition was studied by administering ADC-1, isotype-GRM at different doses (dose 1 and dose 2, wherein dose 2 is 2 times the dose of dose 1) as a single dose (QDx1). Each point on the curve represents the average of five tumors. The error bars depict the standard error of the mean.

[0036] Figure 10: In humanized immunocompetent huCD34+NSG huIL-15 mice, different doses of ADC-1 inhibit the growth of xenografted diffuse large B-cell lymphoma cells (OCI-LY19). A, growth inhibition of xenografted OCI-LY19 tumors by IP administration of isotype mAbs, CD19mAbs and ADC-1. ADC-1 was administered as a single dose (QDx1) at different dose levels (dose 1-3, dose 2 is 3 times the dose of dose 1, and dose 3 is 10 times the dose of dose 1). Each point on the curve represents the average of four or five tumors. The error bars depict the standard error of the mean. B, flow cytometry data of human B cells and human NK cells from tail vein bleeding were obtained at specified time points. Each point on the curve represents the average of four or five animals. The error bars depict the standard deviation of the mean.

[0037] Fig.11 ADC-1 induces excellent anti-tumor activity compared with high-dose CD19 mAb, prednisolone alone, and a combination of CD19 mAb and prednisolone. The growth inhibition of RS4;11 tumors xenografted in CB17 / SCID mice was inhibited by applying a specified treatment. The volume of subcutaneous tumors was plotted according to time after randomization and administration. Fucosylated mAb and ADC-1 were used in this study. Different dose levels (dose 1-3, dose 2 is 10 times the dose of dose 1, and dose 3 is 331 / 3 times the dose of dose 1) were used. Each point on the curve represents the mean of five tumors. The error bars depict the standard error of the mean.

[0038] Figure 12: ADC-1 exhibits antitumor activity in multiple xenograft models of B cell malignancies in vivo compared to prednisolone or isotype GRM controls. AC, DB (A), SUPB15 (B) and RS4; 11 (C) xenograft models were treated with the indicated agents. Various doses were used (dose 1-5, dose 2 was 31 / 3 times the dose of dose 1, dose 3 was 62 / 3 times the dose of dose 1, dose 4 was 10 times the dose of dose 1, and dose 5 was 331 / 3 times the dose of dose 1). As shown in the figure, the mouse strain and CD19 in vitro cell surface copy number of each xenograft tumor model are listed.

[0039] Figure 13: Antitumor efficacy observed in 10 DLBCL patient-derived xenograft (PDX) models. PDX models derived from germinal center B-cell-like (GCB) and non-GCB DLBCL patients were implanted into NOD-SCID mice and when tumors reached approximately 200 mm 3Mice were randomized at the time of randomization. Mice were treated with a single dose of ADC-1 (dose 3, IP) and tumor growth was monitored relative to control-treated PDXs. (A) Tumor growth inhibition is shown as Δ%TGI max=1-(tumor volume of treated group on day X-tumor volume of treated group at randomization) / (tumor volume of control on day X-tumor volume of control at randomization)]*100. The time when the difference between the treated and control groups was the largest was determined. Tumor regression (responders): >105% Δ%TGI max-yes / no response. (B) The number of tumors in the vehicle control group reaching 1000mm calculated by [(tumor volume at time point / tumor volume at randomization)-1]×100 3 The changes in tumor volume at the time points (%).

[0040] Figure 14: Antitumor efficacy observed in R-CHOP relapse PDX model. PDX models derived from DLBCL patients from R-CHOP relapse patients (treated for 4 cycles) were implanted into NOD-SCID mice and when tumors reached approximately 200 mm 3 Mice were randomized at 4 hr. Mice were treated with a single dose of ADC-1 or CD19 antibody at dose 3 (IP), and tumor growth was monitored relative to control-treated PDX. (A) PDX model LY-24-0207 from a 38-year-old male with GCB DLBCL, relapsed after R-CHOP treatment (4 cycles) and had mild stagnation with ibrutinib; (B) PDX model LY-24-0016 from a 59-year-old male with activated B cell (ABC) DLBCL, relapsed after R-CHOP treatment (4 cycles) and resistant to ibrutinib.

[0041] Fig.15 : GRM and ADC-1 induce apoptosis in DLBCL cell lines. Farage, SU-DHL-6, OCI-LY19 were treated with GRM or ADC-1 for the indicated time before cell lysis. The lysates were resolved on SDS-PAGE and immunoblotted with antibodies against the pro-apoptotic regulator BIM and markers of apoptosis induction (cleaved caspase-3, cleaved PARP). Arrows indicate apoptotic cleavage products. GAPDH was used as a loading control.

[0042] Fig.16 : Reduced LC-MS total ion (A280) chromatogram of ADC-1. LC = light chain, LC+2 = light chain + 2 free thiols, DL(X) = drug linker (number of attachments), HC G0 = heavy chain with G0 glycan. 7. Specific implementation methods

[0043] The present disclosure provides antibody drug conjugates (ADCs) comprising a glucocorticoid receptor modulator agonist linked to an anti-CD19 antibody.

[0044] 7.1.CD19 Antibody

[0045] Due to its high expression level on most B cell malignancies (including DLCB, FL, CLL and ALL), CD19 represents an attractive immunotherapy target for cancers of B cell origin. Develop humanized anti-CD19 antibodies that specifically bind to CD19 expressed on the surface of B cells and CD19-positive cancer cells, thereby inhibiting CD19 signaling. In one embodiment, the antibody consists of two heavy chain variable regions and two light chain variable regions. On each variable region, there are three CDRs that allow the antibody to bind to CD19. On two variable chains, there are six different CDRs. In addition, the antibody contains a human heavy chain constant region of a human Fc comprising an immunoglobulin G1 class (IgG1). The anti-CD19 antibodies described herein can be fucosylated or non-fucosylated, and exhibit in vitro functionality, immune safety, and drug-like properties.

[0046] In certain embodiments, the afucosylated antibodies of the present invention have a higher affinity for certain FcγR receptors expressed on immune cells. For example, the afucosylated IgG1 antibody has increased binding to FcγRIIIA expressed on NK cells compared to the fucosylated form of the antibody, resulting in enhanced activity in purified natural killer cells or peripheral blood mononuclear cells (PBMC) ADCC assays. The ADCC activity of anti-CD19 antibodies can be demonstrated using ADCC bioassay techniques known in the art. For example, ADCC reporter gene bioassays in human FcγRIIIa V158 or F158 allele variant reporter gene lines can be used (see Example 3).

[0047] 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 a specific embodiment, the anti-CD19 antibody described herein comprises IgG1. As used herein, the "constant region" of an antibody includes a native constant region, an allotype or a variant.

[0048] The light chain constant region of the anti-CD19 antibody can be a kappa (κ) light chain region or a lambda (λ) region. The λ light chain region can be any known subtype, such as λ1, λ2, λ3 or λ4. In some embodiments, the anti-CD19 antibody comprises a kappa (κ) light chain region.

[0049] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. A monoclonal antibody is derived from a single clone, including any eukaryotic clone.

[0050] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin (eg, a rat or mouse antibody) and a human immunoglobulin constant region (usually selected from a human immunoglobulin template).

[0051] "Humanized" forms of non-human (e.g., mouse) antibodies comprise substantially all of at least one, usually two, variable domains, wherein 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. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence.

[0052] "Human antibodies" include antibodies having human immunoglobulin amino acid sequences, and include antibodies isolated from human immunoglobulin libraries or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous functional immunoglobulins. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0053] The anti-CD19 antibodies of the present disclosure include full-length (intact) antibody molecules.

[0054] Anti-CD19 antibodies can be antibodies whose sequences have been modified to change at least one constant region-mediated biological effector function. For example, anti-CD19 antibodies described herein include antibodies that have been modified to obtain or improve at least one constant region-mediated biological effector function relative to unmodified antibodies, for example, enhancing FcγR interactions (see, e.g., U.S. Patent Application No. 2006 / 0134709, U.S. Patent No. 8,642,292B2) or enhancing the ability of antibody-mediated ADCC. For example, anti-CD19 antibodies of the present disclosure can have a constant region that binds FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and / or FcγRIIIB with greater affinity than the corresponding unmodified constant region. The anti-CD19 antibodies of the present disclosure may be antibodies with modified Fc regions and mediate enhanced ADCC responses, wherein the ADCC response is enhanced relative to antibodies with the same variable regions (i.e., VH and VL) and wild-type IgG1 Fc regions (i.e., wild-type CL, CH1, CH2, and CH3). Anti-CD19 antibodies with high affinity for human CD19 may be ideal for therapeutic and diagnostic purposes. Therefore, the present disclosure contemplates antibodies with high binding affinity for human CD19. In specific embodiments, anti-CD19 antibodies bind to human CD19 with an affinity of about 15nM, 10nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, or 0.1nM. In some embodiments, the antibody binds to human CD19 with an affinity in the range of about 0.1nM to about 10nM.

[0055] In some embodiments, the present invention provides a monoclonal anti-CD19 antibody comprising two sets of six different complementarity determining regions (CDRs), two sets of two different variable regions, two full heavy chains, and two full light chains.

[0056] In some embodiments, the antibody is a recombinant, afucosylated, humanized IgG1κ monoclonal antibody that binds to human CD19.

[0057] In one embodiment, the antibody comprises six CDRs comprising the following sequences:

[0058] CDR-H1: GFTFTTYWIN (SEQ ID NO: 1)

[0059] CDR-H2: NIYPSDSYTNYNQKFKD (SEQ ID NO: 2)

[0060] CDR-H3:EDYYGSSSYYAMDY (SEQ ID NO: 3)

[0061] CDR-L1: KASQDVGTAVA (SEQ ID NO: 4)

[0062] CDR-L2: WASTRHT (SEQ ID NO: 5)

[0063] CDR-L3: QQYSTYPLT (SEQ ID NO: 6)

[0064] In some embodiments, the antibodies of the present disclosure comprise a CDR-H1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR-H2 having an amino acid sequence as shown in SEQ ID NO: 2; a cDR-H3 having an amino acid sequence as shown in SEQ ID NO: 3, a cDR-L1 having an amino acid sequence as shown in SEQ ID NO: 4, a CDR-L2 having an amino acid sequence as shown in SEQ ID NO: 5; and a CDR-L3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0065] In some embodiments, the antibodies of the present disclosure comprise a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO:7: EVQLVESGGGLVQPGGSLRLSCAASGFTFTTYWINWVRQAPGKGLEWIGNIYPSDSYTNYNQKFKDRATLSVDKSKNTAYLQMNSLRAEDTAVYYCTREDYYGSSSYYAMDYWGQGTLVTVSS (SEQ ID NO:7); and a light chain variable region comprising the amino acid sequence as shown in SEQ ID NO:8: AILMTQSPS SLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKVEIK (SEQ ID NO:8).

[0066] In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 9 (the constant region is in normal text; the heavy chain variable domain is Add Line ; CDR is Underline Bold Italic (Disclosed in order of appearance as SEQ ID NOs: 1-3, respectively). N-linked glycosylation sites are indicated by the subscript "g"):

[0067] (full-length sequence disclosed as SEQ ID NO: 9); and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10 (the constant region is normal text; the light chain variable domain Underline ; CDR is Underline Bold Italic (CDR sequences are disclosed in order of appearance as SEQ ID NOs: 4-6, respectively): (Full-length sequence disclosed as SEQ ID NO: 10).

[0068] 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, the heavy chain being the amino acid sequence of SEQ ID NO: 9 truncated with a C-terminal lysine: (Terminal lysine truncated sequence disclosed as SEQ ID NO: 11).

[0069] In one embodiment, the antibody is a CD19 mAb having a heavy chain amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:11 and a light chain amino acid sequence according to SEQ ID NO:10.

[0070] In one embodiment, the heavy chain of an antibody of the present disclosure is encoded by the following nucleotide sequence (full length sequence disclosed as SEQ ID NO: 12):

[0071]

[0072]

[0073] Secretory signal peptide is italicized; final stop codon (TGA) is included; constant region is bold; CDR Underline .

[0074] 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):

[0075]

[0076] Secretory signal peptide is italicized; final stop codon (TGA) is included; constant region is bold; CDR Underline .

[0077] In some embodiments, the antibody comprises a human heavy chain constant region comprising a human CH1, a human hinge, a human CH2, and a human CH3 domain. 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 one embodiment, the Fc is IgG1, and the allotype is z non-a. In one embodiment, the light chain is a kappa light chain.

[0078] In some embodiments, the antibody comprises an IgG1 Fc constant region without fucosylation. Afucosylation can be performed by techniques known in the art. See, for example, Mol Cancer Ther (2020) 19 (5): 1102-1109) and PNAS (2013) 110 (14) 5404-5409. For example, the antibody is produced in a cell line defective in GDP-fucose formation due to, for example, the lack of GDP-mannose 4,6-dehydratase; the antibody is produced in a cell with reduced fucosyltransferase levels; the antibody is produced in a cell with reduced GDP-fucose transporter levels; the antibody is produced in a cell overexpressing β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosamine transferase (GnT-III); or the antibody is produced in a cell expressing bacterial GDP-6-deoxy-D-lyso-4-hexulose reductase (RMD). In one embodiment, the cell used to produce the afucosylated anti-CD19 antibody of the present invention is a CHO cell engineered to express Pseudomonas RMD. The degree of afucosylation of the antibody can be determined by techniques known in the art. Typically, the antibody is 70% or more, 80% or more, 90% or more, or about 99%, or about 100% afucosylated. Preferably, the degree of afucosylation is equal to or greater than 90%. In some embodiments, the antibody is 70% or more, 80% or more, 90% or more, or about 100% afucosylated at position ASN-300 (EU: ASN-297). Afucosylation can be determined via hydrophilic interaction chromatography (HILIC) assay technology, wherein the degree of afucosylation is determined by polarity-dependent separation of fragmented antibodies.

[0079] In one embodiment, the total non-fucosylated glycan species are determined by analyzing the released N-linked glycans by HILIC with fluorescence detection. Glycans are released using peptide N-glycosidase F (PNGase F) and subsequently labeled with a fluorescent tag. Fluorescently labeled N-linked glycans are analyzed by HILIC with fluorescence detection. The percentage of non-fucosylated glycan species is determined based on the sum of the peak areas of all non-fucosylated 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 more are included in the determination of the percentage of non-fucosylated glycan species.

[0080] The present disclosure includes polynucleotide molecules encoding immunoglobulin light chain 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.

[0081] The anti-CD19 antibodies of the present disclosure can be prepared by recombinantly expressing immunoglobulin light chain and heavy chain genes in host cells. In order to recombinantly express antibodies, host cells are transfected with recombinant expression vectors carrying DNA fragments of immunoglobulin light chain and heavy chain encoding antibodies, so that light chain and heavy chain are expressed in host cells and optionally secreted into the culture medium of the host cells, from which antibodies can be recovered.

[0082] In order to generate the polynucleotides encoding such anti-CD19 antibodies, first obtain the DNA fragments encoding the light chain and heavy chain variable regions. These DNAs can be obtained by amplifying and modifying the germline DNA or cDNA encoding the light chain and heavy chain variable sequences, for example, using polymerase chain reaction (PCR).

[0083] Once the DNA fragments encoding the VH and VL segments associated with anti-CD19 antibodies are obtained, these DNA fragments can be further manipulated by conventional recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). The term "operably connected" as used herein refers to connecting two DNA fragments so that the amino acid sequences encoded by the two DNA fragments keep the same reading frame.

[0084] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by conventional 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 the Fab fragment heavy chain gene, the DNA encoding the VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0085] By operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the DNA encoding the separation of the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, e.g., Kabat et al., 1991, "Protein Sequences with Immunological Significance", Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments comprising these regions can be obtained by conventional PCR amplification. The light chain constant region can be a kappa or lambda constant region, but in certain embodiments is a kappa constant region.

[0086] In order to express the anti-CD19 antibody of the present disclosure, the DNA of the light chain and heavy chain of the coding part or full length obtained as described above is inserted into the expression vector so that the gene is operably connected to the transcription and translation control sequence. In this article, the term "operably connected" means that the antibody gene is connected to the vector so that the transcription and translation control sequence in the vector plays its expected function of regulating the transcription and translation of the antibody gene. Expression vector and expression control sequence are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector, or more commonly, two genes are inserted into the same expression vector.

[0087] By conventional methods (for example, connecting complementary restriction sites on antibody gene fragments and vectors, or if there is no restriction site, then flat-end connection is performed) antibody genes are inserted into expression vectors. Before inserting the light chain or heavy chain sequence related to anti-CD19 antibodies, expression vectors may already carry antibody constant region sequences. For example, a method of converting the VH and VL sequences related to anti-CD19 monoclonal antibodies into full-length antibody genes is to insert them into expression vectors encoding heavy chain constant region and light chain constant region, respectively, so that the VH segment is operably connected to the CH segment in the vector, and the VL segment is operably connected to the CL segment in the vector. Additionally or alternatively, recombinant expression vectors can encode signal peptides that promote antibody chains to be secreted from host cells. Antibody chain genes can be cloned into vectors so that signal peptides are connected to the amino termini of antibody chain genes in a consistent reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (that is, a signal peptide from a non-immunoglobulin protein).

[0088] In addition to the antibody chain genes, the recombinant expression vectors of the present disclosure carry regulatory sequences that control the expression of the antibody chain genes in host cells. 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.

[0089] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector of the present disclosure can carry additional sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene helps to select the host cell into which the vector has been introduced. In order to express the light chain and the heavy chain, the expression vector encoding the heavy chain and the light chain is transfected into the host cell by conventional techniques. The various forms of the term "transfection" are intended to include various techniques commonly used to introduce exogenous DNA into eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0090] In certain embodiments, expression of the antibody is performed in eukaryotic cells, such as mammalian host cells, that optimally secrete correctly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing the 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 a DHFR selective marker, e.g., 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 of time 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. The host cell can also be used to produce portions of a complete antibody, such as a Fab fragment or a scFv molecule. It should be understood that variations of the above procedures are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-CD 19 antibody of the disclosure.

[0091] Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that is not necessary for binding to human CD 19. Molecules expressed by such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0092] In order to recombinantly express the anti-CD19 antibody of the present disclosure, the host cell can be co-transfected with two expression vectors of the present disclosure, the first vector encoding the polypeptide of the heavy chain source, and the second vector encoding the polypeptide of the light chain source. The two vectors can contain the same selection marker, or they can each contain a separate selection marker. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used.

[0093] 7.2. Anti-CD19 Antibodies Linked to Glucocorticoid Receptor Modulators

[0094] Provided herein are antibody drug conjugates (ADCs) comprising a glucocorticoid receptor modulator (GRM) linked to an anti-CD19 antibody.

[0095] In certain embodiments, antibody drug conjugates are provided, which contain: (a) an anti-CD19 antibody; and (b) a group of a glucocorticoid receptor modulator of formula (I):

[0096]

[0097] in:

[0098] R 1is hydrogen or -P(=O)(OH)2; and

[0099] Furthermore, wherein the antibody is conjugated to a glucocorticoid receptor modulator via a linker of the formula:

[0100] wherein R is the point of attachment to the linker formed through a cysteine ​​residue of the antibody, providing an -S- group upon attachment. AA1 and AA2 are alanine (A1a), and p and q are 1.

[0101] In certain embodiments, an antibody drug conjugate of formula (II) is provided:

[0102]

[0103] in:

[0104] A is an anti-CD19 antibody; R is the point of attachment to the linker formed by a cysteine ​​residue of the antibody, which provides an -S- group when attached; AA1 and AA2 are alanine (Ala); R 1 is hydrogen or -P(=O)(OH)2 and n is 2 to 10. In embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In embodiments, n is 2, 3, 4, 5, 6, 7 or 8. In another embodiment, n is 1, 2, 3, 4 or 5. In another embodiment, n is 2, 3, 4 or 5. In one embodiment, n is 2, 4, 6 or 8. In another embodiment, n is 2, 4 or 6. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 7. In another embodiment, n is 8.

[0105] In certain embodiments, the anti-CD19 antibody of the ADC comprises a complementary determining region (CDR) as set forth in 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 a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 8. In certain embodiments, the anti-CD19 antibody comprises a heavy chain as set forth in SEQ ID NO: 9 or SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 10, and n is predominantly 2, 4, and 6. In one embodiment, the ADC is ADC-1, wherein the antibody is a CD19 mAb. In certain embodiments, the anti-CD19 antibody heavy chain is encoded by a nucleotide sequence as set forth in SEQ ID NO: 12, and the light chain is encoded by a nucleotide sequence as set forth in SEQ ID NO: 13.

[0106] 7.3. Composition

[0107] The anti-CD19GRM agonist ADC of the present disclosure can be provided as a composition suitable for administration to a subject. In some embodiments, the anti-CD19GRM agonist ADC composition is a pharmaceutical composition comprising the anti-CD19GRM agonist ADC of the present disclosure and a pharmaceutically acceptable carrier.

[0108] According to structural formula II, the ADC composition may include a mixture of ADCs with different integer values ​​of n. The number of GRM agonist drugs connected to the ADC depends on the number of available attachment sites on the antibody. As used herein, "drug-antibody ratio" or "DAR" refers to the molar ratio of the GRM drug to the antibody in the ADC composition.

[0109] In embodiments, the formulation consists essentially of a mixture of ADCs according to formula II having n values ​​of 2, 4, and 6, and the DAR is between 2 and 6.

[0110] 7.4. Usage

[0111] In certain embodiments, the methods described herein relate to treating patients with B cell malignancies such as diffuse large B cell lymphoma (DLBCL), acute lymphoblastic leukemia (ALL), follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL) with CD19GRM agonist ADC. In embodiments, a composition comprising a CD19GRM agonist ADC is administered to a subject in need thereof. In embodiments, the method includes administering ADC, causing a compound of formula III, a compound of formula IV, or a compound of both formula III and formula IV to be delivered to a subject. In embodiments, a compound of formula III, a compound of formula IV, or a compound of both formula III and formula IV is delivered to a B cell malignancy in a subject.

[0112] Administered as a single agent, the CD19-GRM agonist ADC inhibited the growth of subcutaneous xenografts of human tumor cell lines derived from B-cell malignancies, see 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 Fig. 9 ) embodiments.

[0113] 7.5. Methods for generating anti-CD19 ADCs

[0114] In certain embodiments, methods for producing anti-CD19-GRM agonist ADCs are described herein. In a specific embodiment, a method for producing an anti-CD19-GRM agonist ADC is described herein, wherein the antibody is an anti-CD19 antibody comprising two heavy chains and two light chains, each heavy chain consisting of an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 11, and each light chain consisting of an amino acid sequence as shown in SEQ ID NO: 10; wherein the antibody is afucosylated at position ASN-303 (EU: ASN-297); wherein the method comprises the following steps: reducing the antibody with an excess of a reducing agent, and then conjugating the antibody with an excess of a drug linker of formula V.

[0115] 8. Exemplary embodiments

[0116] While various specific embodiments have been illustrated and described, and some are shown below by way of example, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

[0117] 1. An anti-CD19 antibody-drug conjugate, comprising the following structure:

[0118]

[0119] in:

[0120] A is antibody;

[0121] R is the point of attachment of the antibody formed through a cysteine ​​residue of the antibody, which upon attachment provides an -S- group;

[0122] AA1 and AA2 are alanine (Ala);

[0123] R 1 is hydrogen or -P(=O)(OH)2;

[0124] n is an integer from 1 to 10;

[0125] The antibody is an anti-CD19 antibody comprising

[0126] a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and

[0127] A light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein

[0128] CDR-H1 comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1);

[0129] CDR-H2 comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2),

[0130] CDR-H3 comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3);

[0131] CDR-L1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4),

[0132] CDR-L2 comprises the amino acid sequence WASTRHT (SEQ ID NO: 5), and

[0133] CDR-L3 comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6).

[0134] 2. The anti-CD19 antibody-drug conjugate according to embodiment 1, wherein the antibody is an IgG antibody.

[0135] 3. The anti-CD19 antibody-drug conjugate according to embodiment 2, wherein the antibody is an IgG1 antibody.

[0136] 4. The anti-CD19 antibody-drug conjugate according to embodiment 1, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0137] 5. The anti-CD19 antibody-drug conjugate according to embodiment 1, wherein the antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0138] 6. An anti-CD19 antibody-drug conjugate according to embodiment 1, wherein the antibody consists of two identical heavy chains and two identical light chains, wherein each heavy chain consists of the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, and wherein each light chain consists of the amino acid sequence shown in SEQ ID NO: 10.

[0139] 7. The anti-CD19 antibody-drug conjugate of any one of embodiments 1 to 6, wherein the antibody is afucosylated.

[0140] 8. An anti-CD19 antibody-drug conjugate according to embodiment 7, wherein the antibody is 70% or more, 80% or more, 90% or more, or about 99% or about 100% afucosylated.

[0141] 9. The anti-CD19 antibody-drug conjugate of embodiment 7 or embodiment 8, wherein the afucosylation is at position ASN-300 (EU: ASN-297).

[0142] 10. A pharmaceutical composition comprising the anti-CD19 antibody-drug conjugate according to any one of embodiments 1 to 9 and a pharmaceutically acceptable carrier.

[0143] 11. A method of treating a B-cell malignancy, the method comprising administering to a patient in need thereof an antibody-drug conjugate according to any one of embodiments 1 to 9.

[0144] 12. The anti-CD19 antibody-drug conjugate of any one of embodiments 1 to 9, wherein n is 2, 4 or 6.

[0145] 13. An anti-CD19 antibody-drug conjugate according to any one of embodiments 1 to 9, wherein n is 2.

[0146] 14. The anti-CD19 antibody-drug conjugate of any one of embodiments 1 to 9, wherein n is 4.

[0147] 15. The anti-CD19 antibody-drug conjugate of any one of embodiments 1 to 9, wherein n is 6.

[0148] 16. An anti-CD19 antibody, comprising

[0149] a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain; and

[0150] A light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein

[0151] CDR-H1 comprises the amino acid sequence GFTFTTYWIN (SEQ ID NO: 1);

[0152] CDR-H2 comprises the amino acid sequence NIYPSDSYTNYNQKFKD (SEQ ID NO: 2),

[0153] CDR-H3 comprises the amino acid sequence EDYYGSSSYYAMDY (SEQ ID NO: 3);

[0154] CDR-L1 comprises the amino acid sequence KASQDVGTAVA (SEQ ID NO: 4),

[0155] CDR-L2 comprises the amino acid sequence WASTRHT (SEQ ID NO: 5), and

[0156] CDR-L3 comprises the amino acid sequence QQYSTYPLT (SEQ ID NO: 6).

[0157] 17. An anti-CD19 antibody according to embodiment 16, wherein the antibody is an IgG antibody.

[0158] 18. The anti-CD19 antibody of embodiment 17, wherein the antibody is an IgG1 antibody.

[0159] 19. An anti-CD19 antibody according to embodiment 16, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0160] 20. The anti-CD19 antibody of embodiment 16, wherein the antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0161] 21. An anti-CD 19 antibody according to any one of embodiments 16 to 20, wherein the antibody is afucosylated.

[0162] 22. An anti-CD 19 antibody according to embodiment 21, wherein the antibody is 70% or more, 80% or more, 90% or more, or about 99% or about 100% afucosylated.

[0163] 23. The anti-CD 19 antibody of embodiment 21 or embodiment 22, wherein the afucosylation is at position ASN-300 (EU: ASN-297).

[0164] 24. An anti-CD19 antibody according to embodiment 16 or 19, wherein the antibody 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, a scFv-Fc antibody, Fab, Fab′, (Fab′)2, Fv or scFv.

[0165] 25. An anti-CD 19 antibody according to embodiment 24, wherein the antibody is afucosylated.

[0166] 26. An anti-CD 19 antibody according to embodiment 25, wherein the antibody is 70% or more, 80% or more, 90% or more, or about 99% or about 100% afucosylated.

[0167] 27. The anti-CD 19 antibody of embodiment 25 or embodiment 26, wherein the afucosylation is at position ASN-300 (EU: ASN-297).

[0168] 28. An anti-CD19 antibody-drug conjugate, comprising the following structure:

[0169]

[0170] in:

[0171] A is antibody;

[0172] R is the point of attachment of the antibody formed through a cysteine ​​residue of the antibody, which upon attachment provides an -S- group;

[0173] AA1 and AA2 are alanine (Ala);

[0174] R 1 is -P(=O)(OH)2;

[0175] n is an integer from 2 to 8;

[0176] The antibody is an anti-CD19 antibody comprising

[0177] Two heavy chains and two light chains, each heavy chain consists of the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, and each light chain consists of the amino acid sequence shown in SEQ ID NO: 10.

[0178] wherein the antibody is afucosylated at position ASN-303 (EU: ASN-297).

[0179] 29. An anti-CD19 antibody according to embodiment 28, wherein n is 2.

[0180] 30. An anti-CD19 antibody according to embodiment 28, wherein n is 4.

[0181] 31. An anti-CD19 antibody according to embodiment 28, wherein n is 6.

[0182] 32. An anti-CD19 antibody according to embodiment 28, wherein n is 8.

[0183] 33. One or more polynucleotides encoding the anti-CD19 antibody according to embodiment 16.

[0184] 34. A method for producing an anti-CD19 antibody-drug conjugate comprising the structure:

[0185]

[0186] in:

[0187] A is antibody;

[0188] R is the point of attachment of the antibody formed through a cysteine ​​residue of the antibody, which upon attachment provides an -S- group;

[0189] AA1 and AA2 are alanine (Ala);

[0190] R 1 is -P(=O)(OH)2;

[0191] n is an integer from 2 to 8;

[0192] The antibody is an anti-CD19 antibody comprising

[0193] two heavy chains and two light chains, each heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, and each light chain consisting of the amino acid sequence shown in SEQ ID NO: 10;

[0194] wherein the antibody is afucosylated at position ASN-303 (EU: ASN-297);

[0195] The method comprises the following steps: reducing the antibody with an excess of a reducing agent, and then conjugating the antibody with an excess of the drug linker comprising the following structure:

[0196]

[0197] 9. Examples

[0198] The following examples highlight certain features and characteristics of exemplary embodiments of the antibodies and binding fragments described herein and are provided for purposes of illustration.

[0199] 9.1. Example 1: Production of CD19 Antibodies

[0200] Hybridoma technology was used to isolate CD19 antibodies consisting of the complementary determining regions (CDRs) of mouse antibody clone 4B12 (mouse anti-human CD19). These CDR sequences (SEQ ID NOs: 1-6) were cloned into human IgG1 heavy chains and human kappa light chain constant region domains with 9 mouse variable heavy chain framework reversion mutations and 2 mouse variable light chain framework reversion mutations to generate H chain (SEQ ID NO: 9) and L chain (SEQ ID NO: 10) sequences. Humanized CD19 antibodies (CD19 mAbs) retain the CD19 binding potency and selectivity of the parental mouse hybridoma clone 4B12.

[0201] Chinese hamster ovary (CHO) cells are transfected by electroporation with an expression vector (pCD-CD19 antibody) designed to express CD19 antibodies. Cells are grown in a chemically defined culture medium so that cells expressing DHFR can be selected from expression vectors and CD19 antibodies. Cells are grown and cloned growths are screened for anti-CD19 antibody expression. Based on screening, stable transfectants are identified as parental cell lines for further development.

[0202] The stable transfectant was then transfected with an additional vector (pYH9neo-RMD, which expresses Pseudomonas GDP-6-deoxy-D-lyso-4-hexulose reductase (RMD)) by electroporation. The expression vector enables the production of afucosylated CD19 antibody (greater than or equal to 90% afucosylation) in a cell line defective in GDP-fucose formation due to, for example, a defect in GDP-mannose 4,6-dehydratase. Clonal cells were screened for growth and expression of afucosylated CD19 antibody. The final subclone was selected and a master cell bank was generated.

[0203] 9.2. Example 2: Synthesis of ADC-1 (a CD19 GRM agonist ADC)

[0204] 9.2.1. Overview

[0205] By conjugating the anti-CD19 antibody of Example 1 (to a linker drug containing a GRM of Formula I, wherein R 1 is a phosphate group) to form a CD19GRM agonist ADC. Phosphate-GRM payload of formula (III):

[0206]

[0207] Conjugated to CD19 antibody via an alanine-alanine cleavable linker. After administration of the ADC, the phosphate-GRM payload of Formula III is converted to the GRM of Formula IV:

[0208]

[0209] The conjugated material was treated using hydrophobic interaction chromatography (HIC) to substantially reduce antibodies containing zero conjugated linker-drug and ADCs containing eight GRM molecules per antibody, resulting in a final drug product comprising a mixture of ADCs containing predominantly two, four, or six GRM molecules, with DARs between two and six.

[0210] The analytical procedure used to generate ADC-1, a CD19 GRM agonist ADC, is described in detail below.

[0211] 9.2.2. Small molecule analysis procedures

[0212] Unless otherwise stated, all data were collected using a Varian Mercury Plus 400 MHz, Agilent MR400 MHz, or Bruker AVIII 300, 400, 500, or 600 MHz instrument. 1 H and 13C NMR (nuclear magnetic resonance) data; chemical shifts are expressed in parts per million (ppm). High performance liquid chromatography (HPLC) and LCMS analytical data are described in detail within the experiments or refer to the conditions listed in Table 1.

[0213]

[0214]

[0215] 9.2.3. Synthesis of precursor molecules

[0216] 9.2.3.1. (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, Synthesis of 11a,12,12a,12b-dodecahydro-4H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-4-one become

[0217] (GRM of Formula IV)

[0218]

[0219] Step 1: Synthesis of 4-(bromomethyl)benzaldehyde. Diisobutylaluminum hydride (153mL, 153mmol, 1M in toluene) was added dropwise to a 0°C solution of (4-bromomethyl)benzonitrile (20g, 102mmol) in toluene (400mL) over 1 hour. Two additional reactions were set up as described above. All three reaction mixtures were combined for purification. The combined reaction mixtures were quenched with 10% aqueous HCl solution (1.5L), extracted with dichloromethane (3×500mL), dried (Na2SO4), and the solvent was removed under reduced pressure. Purification by column chromatography (silica gel) eluted with 10:1 petroleum ether / ethyl acetate gave the title compound (50g, 82% yield). 1 H NMR (400MHz, CDCl3) δ10.02 (s, 1H), 7.91-7.82 (m, 2H), 7.56 (d, J = 7.9Hz, 2H), 4.55-4.45 (m, 2H).

[0220] Step 2: Synthesis of 3-(4,4,5,5-tetramethyl-1,3,2-di-oxaborolan-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′-bis(1,3,2-dioxaborolan) (94 g, 372 mmol), potassium acetate (45.6 g, 465 mmol), 2-dicyclohexylphosphine-2′,4′,6′-tri-isopropyl-1,1′-biphenyl (8.07 g, 13.95 mmol), tris(dibenzylideneacetone)dipalladium(0) (8.52 g, 9.30 mmol). The resulting mixture was then heated at 80° C. for 4 hours under nitrogen. Other reactions were 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). 1 H NMR (400MHz, CDCl3) δ7.23-7.13 (m, 3H), 6.80 (d, d=7.5Hz, 1H), 3.82-3.38 (m, 2H), 1.34 (s, 12H).

[0221] 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), washed with 0.1 N HCl aqueous solution (3×2 L), brine (3 L), dried (Na2SO4), filtered and concentrated under reduced pressure to give the title compound (50 g, 57% yield). 1 H NMR (400MHz, CDCl3) δ7.63 (br m, 2H), 7.48 (d, J = 7.1Hz, 1H), 7.37-7.28 (m, 1H), 1.52 (s, 9H), 1.34 (s, 12H).

[0222] 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 (Na 2 SO 4 ) and the solvent 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) δ9.95 (s, 1H), 7.78 (d, J=7.9Hz, 2H), 7.33 (d, J=7.9Hz, 2H), 7. 27-7.13 (m, 3H), 6.82 (d, J=7.1Hz, 1H), 6.47 (br.s., 1H), 4.00 (s, 2H), 1.48 (s, 9H).

[0223] 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]phenanthrene-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 40% HBF4 aqueous solution (440 mL) and the mixture was stirred at 25°C for 48 hours. After the reaction was complete, 2 L of water was added and the solid was collected by filtration. The solid was washed with water (1 L) and then methanol (200 mL) to give the title compound (11 g, 60.3% yield). 1H NMR (400MHz, DMSO-d6) δ7.25 (d, J=10.1Hz, 1H), 6.28 (d, J=10.1Hz, 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=19.4Hz, 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).

[0224] 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]phenanthrene-3-one (4.4 g, 10.67 mmol) and MgSO4 (6.42 g, 53.3 mmol) in acetonitrile (100 mL) was stirred at 20° C. for 1 hour. 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 keeping the internal temperature below room temperature using an ice bath. After addition, the mixture was stirred at 20 °C for 2 hours. Three other reactions were set up as described 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 (400MHz, DMSO) δ7.34 (d, J=7.7Hz, 2H), 7.24 (dd, J=16.2, 9.0Hz, 3H), 6.88 (t, J=7.8Hz, 1H), 6.43-6 .33 (m, 3H), 6.30 (d, J = 10.2Hz, 1H), 6.13 (s, 1H), 5.75-5.55 (m, 1H), 5.52 (d, J = 4.1Hz, 1H), 5.44 (s, 1H), 5.11 (t, J=6.0Hz, 1H), 4.94 (s, 3H), 4.51 (dd, J=19.6, 6.5Hz, 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: Gilson 281 semi-preparative HPLC system; mobile phase A: formic acid / H2O=0.01% v / v; B: acetonitrile; column: Luna C18150*255 microns; flow rate: 25 mL / min; monitoring wavelengths: 220 nm and 254 nm.

[0225]

[0226] 9.2.3.2. 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[l,2-d][1.3]dioxole-8b- Synthesis of 2-oxoethyl ester

[0227] (Phosphate-GRM of Formula III)

[0228]

[0229]

[0136] Phosphorus oxychloride (6.86 g, 27.2 mmol) was added dropwise to a -40 °C 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-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 p-toluenesulfonic acid (0.938 g, 5.45 mmol) in tetrahydrofuran (30 mL). The mixture was stirred at -40 ° C for 2 hours. Three additional reactions were set as described above. The combined reaction mixture was quenched with water (450 mL) at -40 ° C, warmed to 0 ° C and adjusted to pH about 7 by adding saturated NaHCO The aqueous solution was adjusted to pH about 7. Because a large amount of liquid volume filled the flask, the suspension was filtered at pH about 6. The filtrate was then adjusted to pH 7 and rinsed through the filtered solid. The filtrate was acidified back to pH 3 by adding 1M HCl aqueous solution (200 mL) to obtain a suspension. The mixture was filtered and the combined solid was rinsed twice with mother liquor and collected. The crude solid was dried under vacuum and purified by preparative HPLC on Nano-micro Kromasil C183 micron column (100 × 40 mm). At a flow rate of 50 mL / minute, a gradient (0-5.0 minute 20% A, 5.0-25.0 minute linear gradient 20-50% A, kept for 5 minutes) of acetonitrile (A) and water (B) containing 1% trifluoroacetic acid was used. The purified fractions were adjusted to pH about 5 and lyophilized to give the title compound as a white solid (3.50 g, 23% yield). LCMS (Method b, Table 1) Rt = 0.707 min; m / z 686.2 (M+H + ). 1H NMR (400MHz, DMSO-d6) δ7.35 (d, J=7.9Hz, 2H), 7.26 (td, J=7.3, 6.2, 3.1Hz, 3H), 7.07 (t, J=7.9Hz, 1H), 6.7 3-6.60 (m, 3H), 6.30 (dd, J=10.2, 1.9Hz, 1H), 6.12 (d, J=2.4Hz, 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, 3.6Hz, 1H), 1.72 (q, J=9.3, 8.1Hz, 3H), 1.60-1.41 (m, 1H), 1.50 (s, 3H), 0.88 (s, 3H). 19 F NMR (376MHz, DMSO-d6) delta -73.76, -164.99, -186.36. 31 P NMR (162MHz, DMSO-d6) δ-1.17.

[0230] 9.2.3.3. (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] Synthesis of [1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propionamide (drug of formula VI) (Plug)

[0231]

[0232] 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.

[0233] 2,6-Lutidine (0.5 mL, 4.29 mmol) was added to (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,7,8,8 a,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-butyloxycarbonyl)amino)propionamido)propanoic acid [CAS No. 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) were suspended in tetrahydrofuran (5.5 mL) at room temperature. After 16 hours, the reaction was diluted with EtOAc (40 mL) and washed with 1N aqueous HCl (2×20 mL), saturated aqueous NaHCO 3 (20 mL), brine (20 mL), dried (Na 2 SO 4 ), filtered and the solvent removed under reduced pressure. Purification by chromatography (silica, 40 g) eluting with a gradient of 0-10% MeOH / DCM gave the title compound (0.648 g, 92% yield) as an off-white solid. LCMS (Method c, ESI, Table 1) Rt=0.89 min; m / z=848.6 (M+H + ). 1H NMR (600MHz, DMSO-d6) δ9.82 (s, 1H), 7.94 (d, J = 7.3Hz, 1H), 7.46 (d, J = 7.9Hz, 1H), 7.3 8(t, J=2.0Hz, 1H), 7.38-7.33(m, 2H), 7.26(dd, J=10.2, 1.5Hz, 1H), 7.26-7.22(m, 2H), 7.19 (t, J=7.9Hz, 1H), 6.95 (d, J=7.4Hz, 1H), 6.91 (dt, J=7.7, 1.2Hz, 1H), 6.29 (dd, J=1 0.1, 1.9Hz, 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=5.9Hz, 1H), 4.94 (d, J=5.2Hz, 1H), 4.51 (dd, J=19.4, 6.1Hz, 1H), 4.35 (t, J= 7.1Hz, 1H), 4.23-4.16 (m, 2H), 3.97 (t, J=7.2Hz, 1H), 3.88 (s, 2H), 2.65 (ddt, J=28.2, 1 5.3, 7.9Hz, 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=7.1Hz, 3H), 1.20-1.15 (m, 3H), 0.86 (s, 3H).

[0234] 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

[0235] Trifluoroacetic acid (2.0 mL, 26.0 mmol) was added to ((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 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 NaHHCO3 (3 x 20 mL), saturated aqueous NaBr (20 mL), dried (Na2SO4), filtered and the solvent removed under reduced pressure. The residue was triturated with MTBE / heptane and the solvent removed to give the title compound which was used in the next step without further purification. LCMS (Method c, ESI, Table 1) Rt = 0.72 min; m / z 748.5 (M+H + ).

[0236] 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.

[0237] 2,6-Dimethylpyridine (0.2 mL, 1.717 mmol) was added to (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 (0.286 g, 0.382 mmol) and bromoacetic acid N-hydroxysuccinimide ester (0.414 g, 1.753 mmol) in dimethylformamide (4 mL) at room temperature. The reaction was complete in 20 minutes as monitored by LCMS. A second reaction of the same size was set up. The two reactions were combined and purified by preparative HPLC on a Phenomenex C18(2) 10 micron column (250×50 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, hold for 5 min). 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) Rt = 0.82 min; m / z 868.4, 870.3 (M+H) + . 1HNMR (600MHz, DMSO-d6) δ9.82 (s, 1H), 8.45 (d, J = 7.3Hz, 1H), 8.20 (d, J = 7.2Hz, 1H), 7 .46 (dd, J=8.4, 2.2Hz, 1H), 7.40 (t, J=1.9Hz, 1H), 7.38-7.33 (m, 2H), 7.28-7.19 (m, 4H ), 7.19 (d, J=7.9Hz, 1H), 6.91 (dt, J=7.7, 1.3Hz, 1H), 6.29 (dd, J=10.2, 1.9Hz, 1H), 6 .12 (d, J=2.0Hz, 1H), 5.64 (dddd, J=48.5, 11.4, 6.8, 1.9Hz, 1H), 5.51 (d, J=4.4Hz, 1H) , 5.45 (s, 1H), 4.94 (d, J = 5.3Hz, 1H), 4.51 (d, J = 19.4Hz, 1H), 4.40-4.33 (m, 1H), 4.31 (p, J=7.2Hz, 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, 5.7Hz, 1H), 2.04 (dt, J=13.7, 3.7Hz, 1H), 1.77-1.63 (m, 3H), 1.57 -1.50 (m, 1H), 1.50 (s, 3H), 1.27 (d, J=7.1Hz, 3H), 1.21 (d, J=7.1Hz, 3H), 0.86 (s, 3H).

[0238] 9.2.3.4. 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-f4-dihydrogen phosphate f3-((S)-2-((S)-2-(2-bromoacetamido)propionamido)propionamido)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′, Synthesis of 1′:4,5]-indeno[1.2-d][1.3]dioxol-8b-yl)-2-oxoethyl ester (drug linker of formula V)

[0239]

[0240] 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.

[0241] 2,6-Lutidine (1.2 mL, 10.30 mmol) was added to (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,7,8,8 a,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 No. 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) in tetrahydrofuran (15 mL) at room temperature. After 15 h, the mixture was diluted with EtOAc (100 mL) and washed with 1N aqueous HCl (2 x 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) Rt = 0.98 min, m / z 970.6 (M+H + ). 1H NMR (400MHz, DMSO-d6) δ9.83 (s, 1H), 8.06 (d, J=7.3Hz, 1H), 7.91-7.84 (m, 2 H), 7.71 (t, J=7.8Hz, 2H), 7.53 (d, J=7.4Hz, 1H), 7.49-7.13 (m, 12H), 6.91 ( dt, J=7.7, 1.4Hz, 1H), 6.29 (dd, J=10.2, 1.9Hz, 1H), 6.14 (q, J=1.6Hz, 1H), 5.73-5.54 (m, 1H), 5.52 (dd, J=4.4, 1.8Hz, 1H), 5.44 (s, 1H), 5.10 (t, J=5.9 Hz, 1H), 4.95 (d, J=4.8Hz, 1H), 4.51 (dd, J=19.5, 6.2Hz, 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, 1 H), 2.35-2.18(m, 2H), 2.08-1.96(m, 1H), 1.77-1.6l(m, 3H), 1.60-1.49(m, 1H), 1.50 (s, 3H), 1.27 (d, J=7.1Hz, 3H), 1.23 (d, J=7.1Hz, 3H), 0.86 (s, 3H).

[0242] Step 2: ((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-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)carbamic acid (9H-fluoren-9-yl)methyl ester.

[0243] Di-tert-butyl N,N-diethylphosphoramidite (1.8 mL, 6.02 mmol) was added to ((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,11 a,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)carbamic acid (9H-fluoren-9-yl)methyl ester (1.98 g, 2.041 mmol) and tetrazole (0.45 M in acetonitrile) (18 mL, 8.10 mmol) at room temperature. After 50 minutes, the reaction was treated with hydrogen peroxide (30% aqueous solution, 1.2 mL, 11.75 mmol). After 10 minutes, the reaction was treated with 1 M NaS2O3 aqueous solution (80 mL) to quench any unreacted hydrogen peroxide [Caution: exothermic!]. The mixture was extracted with EtOAc (3×60 ml). The combined organic layers were washed with brine (50 mL), dried (Na2SO4), 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 desired product (1.85 g, 78% yield) as a white foam. LCM (Method d, ESI, Table 1) Rt = 1.12 min, m / z 1162.7 (M+H + ).

[0244] Step 3: Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-aminopropionamido)propionamido)benzyl)phenyl)-2,6b-di-fluoro-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.

[0245] Piperidine (1.0 ml, 10.10 mmol) was added to ((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-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)carbamic acid (9H-fluoren-9-yl)methyl ester (1.854 g, 1.595 mmol) in tetrahydrofuran (16 mL) at room temperature. After 25 minutes, the solvent was removed under reduced pressure. The residue was repeatedly treated with toluene (3×30 mL), and the toluene was removed under reduced pressure after each treatment to ensure removal of piperidine. Assuming a yield of 100%, the crude product was used in the next step without further purification. LCMS (Method d, ESI, Table 1) Rt=1.11 min, m / z 940.7 (M+H + ).

[0246] Step 4: Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propionamido)propionamido)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.

[0247] 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 minutes 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-aminopropionamido)propionamido)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). The solution was transferred to a vial containing activated bromoacetic acid. Due to incomplete reaction, a second batch 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) and stirred for 70 minutes, then merged with the reaction mixture. After 15 minutes, the reaction was diluted with EtOAc (60 mL) and washed with water (2 × 20 mL), dried (Na2SO4), filtered and the solvent was removed under reduced pressure. Purified by chromatography (silicon dioxide, 80 g) with a gradient elution of 0-10% MeOH / DCM to obtain a product (0.760 g, 45% yield) as a white solid. LCMS (method c, ESI, Table 1) Rt = 1.03 minutes, m / z 1060.6, 1062.1 (M+H + ). 1H NMR (400MHz, DMSO-d6) δ9.83 (s, 1H), 8.47 (d, J = 7.3Hz, 1H), 8.22 (d, J = 7.2Hz, 1H ), 7.49-7.43(m, 1H), 7.41(t, J=1.9Hz, 1H), 7.35(d, J=8.2Hz, 2H), 7.32-7.23(m , 3H), 7.19 (t, J=7.8Hz, 1H), 6.91 (dt, J=7.7, 1.4Hz, 1H), 6.30 (dd, J=10.2, 1.9H z, 1H), 6.12 (d, J=2.2Hz, 1H), 5.74-5.55 (m, 2H), 5.56 (s, 1H), 5.03-4.90 (m, 2H) , 4.65 (dd, J=18.0, 9.3Hz, 1H), 4.34 (dt, J=15.6, 7.2Hz, 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 =13.2Hz, 1H), 1.72 (td, J = 13.9, 4.2Hz, 2H), 1.67-1.52 (m, lH), 1.49 (s, 3H), 1.4 2 (s, 9H), 1.42 (s, 9H), 1.27 (d, J=7.1Hz, 3H), 1.20 (d, J=7.0Hz, 3H), 0.89 (s, 3H).

[0248] Step 5: Synthesis of 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propionamido)propionamido)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.

[0249] Trifluoroacetic acid (2.5 mL, 32.4 mmol) was added to 2-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-bromoacetamido)propionamido)propionamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-di-butylphosphonate. A room temperature solution of 6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8b(H-naphtho[2′,1′:4,5]-indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl ester (0.760 g, 0.716 mmol) in dichloromethane (5 mL) was stirred in an open vessel for 15 minutes and 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 (250×50 mm). At a flow rate of 90 mL / min, a gradient of acetonitrile (A) and water containing 1% trifluoroacetic acid (B) was used (0-5.0 min 15% A, 5.0-20.0 min linear gradient 15-95% A, hold for 5 min). The product fractions were freeze-dried to give the title compound (0.291 g, 43% yield) as a white solid. LCMS (Method c, APCI, Table 1) Rt = 0.84 min, m / z 948.4, 950.4 (M+H + ). 1H NMR (600MHz, DMSO-d6) δ9.83 (s, 1H), 8.46 (d, J = 7.4Hz, 1H), 8.21 (d, J = 7.3Hz, 1H) , 7.46 (ddd, J=8.2, 2.2, 1.1Hz, 1H), 7.39 (t, J=1.9Hz, 1H), 7.38-7.33 (m, 2H), 7.29 -7.23 (m, 3H), 7.19 (t, J=7.8Hz, 1H), 6.91 (dt, J=7.7, 1.3Hz, 1H), 6.30 (dd, J=10. 1, 1.9Hz, 1H), 6.13 (dt, J=2.6, 1.3Hz, 1H), 5.64 (dddd, J=48.3, 11.1, 6.4, 1.9Hz, 2 H), 5.53 (s, 1H), 4.96-4.89 (m, 2H), 4.59 (dd, J=18.1, 8.4Hz, 1H), 4.34 (dp, J=23. 2, 7.1Hz, 2H), 4.21 (d, J=9.0Hz, 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, 6.3Hz, 2H), 1.68 (s, 1H), 1.57-1. 49 (m, 1H), 1.49 (s, 3H), 1.27 (d, J=7.1Hz, 3H), 1.21 (d, J=7.1Hz, 3H), 0.88 (s, 3H).

[0250] 9.2.4. Conjugation and analytical methods

[0251] 9.2.4.1. Small molecule analysis procedures

[0252] High performance liquid chromatography (HPLC) and LCMS analytical data are described in detail within the experiments or refer to the conditions listed in Table 3.

[0253]

[0254] 9.2.4.2.CD19 phosphorylated GRM agonist ADC

[0255] 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 water for injection (WFI) solution) at about 4° C. under nitrogen overnight. The reduced antibody was then conjugated to a drug linker of Formula V:

[0256]

[0257] (6.0 eq., 6.92 g, 93.2% w / w%, 650 g of a solution in dimethylacetamide (DMA)) was added at about 4 °C, then warmed to 20 °C and kept for 6-8 hours until judged complete. The conjugation was quenched by adding N-acetylcysteine ​​(20 eq., 3.71 g, 100 mM WFI solution). The product solution was divided into 2 portions, each of which was subsequently diluted to about 13 L with WFI, and the pH was subsequently adjusted to about 8.5 with NaOH as the loading solution. Each loading solution was purified.

[0258] 9.2.4.3. Mass spectrometry

[0259] Before MS analysis, the ADC samples were completely reduced. The mass spectrometry conditions used were as follows: HPLC column = Waters BioResolve RP mAb Polyphenyl 450A 2.7um, 150×2.1mm; Mobile phase A: water containing 0.1% difluoroacetic acid; Mobile phase B: acetonitrile containing 0.1% difluoroacetic acid Flow rate: 0.4mL / min; Gradient: start 20% B, 0 to 12 minutes 20-45% B, 14 to 16 minutes 45-60% B, 16 to 18 minutes 60-100% B, re-equilibrate 20% B for 4 minutes; Column temperature: 80°C; MS ionization source: ESI.

[0260] Generate reduced reversed-phase (A280) chromatograms and deconvoluted mass spectra for each subunit. Fig.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.

[0261] 9.2.4.4.CD19 dephosphorylating GRM agonist ADC

[0262] 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 with PBE (125 mM phosphate, 6 mM EDTA pH 7.3) buffer and partially reduced with TCEP (2.56 eq, 0.0141 g, 4.93 mM WFI solution) at about 4° C. under nitrogen overnight. The reduced antibody was then conjugated to a drug linker of Formula VI:

[0263]

[0264] Add (6.1 eq., 0.102 g, 90.2% w / w%, 11 g solution in DMA) at about 4 °C, then warm to 20 °C and hold for 6-8 hours until judged complete. Conjugation is quenched by adding N-acetylcysteine ​​(10 eq., 0.031 g, 100 mM WFI solution). The product solution is salified with 0.8 M ammonium sulfate, 25 mM sodium phosphate pH 7.1 (126 g) to prepare a loading solution for purification. Purify the loading solution.

[0265] 9.3. Example 3: Characterization of Antibodies and ADCs

[0266] Characterize the binding and cellular activity of the CD19 phosphorylation GRM agonist ADC (ADC-1) and its parent non-fucosylated antibody (also referred to as CD19 mAb) of Section 8.2.4.2 of Example 2. ADC-1 and CD19 mAb bind to CD19 endogenously expressed on the surface of multiple B cell lymphoma cell lines. ADC-1 triggers effective anti-proliferative effects and ADCC activity against multiple B cell malignancy cell lines. In addition, ADC-1 activates NFAT ADCC reporter cells expressing F158 or V158 FcγRIIIa variants with similar efficacy. Although ADC-1 exhibits negligible binding to mouse or rat CD19, it exhibits high affinity binding to human and cynomolgus monkey (cyno) CD19 and activated GRE reporter cell lines expressing human or cyno CD19.

[0267] Materials and methods

[0268] 9.3.1.1. Antibodies, proteins and compounds

[0269] Humanized anti-CD19 antibody (CD19 mAb) is made afucosylated with ProBiogen technology. ADC-1 is prepared by conjugating CD19 mAb to a drug linker of formula V. GLP materials for CD19 mAb and ADc-1 are used for all experiments. The negative control tested includes afucosylated anti-tetanus toxoid antibody AB095 (isotype mAb) and its ADC (isotype-GRM), and its ADC is composed of AB095 and a drug linker of formula V. The compounds analyzed include GRM, dexamethasone (Clinigen, catalog number 00641-0367-25) and prednisolone (Clinigen, catalog number 44523-0182-08) of formula IV.

[0270] 9.3.1.2. Cell culture

[0271] Cancer cell lines Raji (Burkitt's lymphoma; ATCC, catalog number CCL-86), SUP-B15 (acute lymphoblastic leukemia; ATCC, catalog number CRL-1929), RS4;11 (acute lymphoblastic leukemia; ATCC, catalog number CRL-1873), SU-DHL-6 (diffuse large B-cell lymphoma; ATCC, catalog number CRL-2959) and HCT116 (colorectal carcinoma; ATCC, catalog number CCL-247) were cultured in RPMI-1640 medium (Thermo Fisher Scientific, catalog number A1049101) supplemented with 10% FBS (Thermo Fisher Scientific, catalog number 26140079). The cancer cell line OCI-LY19 (diffuse large B-cell lymphoma DSMZ, catalog number ACC 528) is cultured in IMDM medium with 10% human serum (Sigma-Aldrich, catalog number P2918-100ML) sterile filtered from platelet-poor human plasma. 293T (ATCC, catalog number CRL-3216) cells are cultured in DMEM supplemented with 10% FBS. All cells are cultured in a humidified chamber at 37°C and 5% CO2.

[0272] 9.3.1.3. Generation of engineered cell lines

[0273] The K562GRE luciferase reporter cell line expressing human CD19 was generated at AbbVie. Briefly, K562 cells were seeded at 500,000 cells / well into 6-well culture dishes (Costar, catalog number 3516) with 2 mL of complete growth medium [RPMI 1640 supplemented with L-glutamine (Thermo Fisher Scientific, catalog number 11835-030), 10% FBS (Thermo Fisher Scientific Inc., catalog number 26140-079), 1% sodium pyruvate (Thermo Fisher Scientific Inc., catalog number 11360-070) and 1% MEM NEAA (Thermo Fisher Scientific Inc., catalog number 111140-50)] at 37°C and 5% CO2 for 24 hours. The next day, 1.5 μg pGL4.36 [Luc2P / MMTV / Hygro] (Promega catalog number E316) and 3 μL PLUS reagent (Thermo Fisher Scientific, catalog number 10964-021) were diluted in 244 μL Opti-MEM (Thermo Fisher Scientific, catalog number 31985-070) and incubated at room temperature for 15 minutes. After incubation, the diluted DNA solution was pre-incubated with 1: 1 Lipofectamine LTX solution (Thermo Fisher Scientific, catalog number 94756) (13.2 μL + 256.8 μL Opti-MEM) and incubated at room temperature for 25 minutes to form DNA-Lipofectamine LTX complex. After incubation, 500 μL DNA-Lipofectamine complex was added directly to the well containing the cells. K562 cells were transfected at 37 ° C and 5% CO2 for 24 hours. After incubation, cells were washed with 3 mL of PBS and selected for two weeks with complete growth medium containing 125 μg / mL of hygromycin B (Thermo Fisher Scientific, catalog number 10687-010).

[0274] K562 pGL4.36[Luc2P / MMTV / Hygro] cells were harvested and seeded at 250,000 cells per well in a 15 mL conical tube (Costar, catalog number 3516) with 1 mL complete growth medium (RPMI+L-Glu, 10% FBS, 1% sodium pyruvate, and 1% MEM NEAA). 3 μg human CD19 (Origene, #RC230267) and 3 μL PLUS reagent (Thermo Fisher Scientific, catalog number 10964-021) were diluted in 244 μL Opti-MEM (Thermo Fisher Scientific, catalog number 31985-070) and incubated at room temperature for 5 minutes. After incubation, the diluted DNA solution was incubated with 1: 1 Lipofectamine LTX solution (Thermo Fisher Scientific, catalog number 94756) (11 μL + 239 μL Opti-MEM) in advance and incubated at room temperature for 15 minutes to form DNA-Lipofectamine LTX complex. Subsequently, 500 μl DNA-Lipofectamine complex was directly added to the conical tube containing the cells. Cells and DNA-lipofectamine were mixed and inoculated into 6-well culture dishes (Costar, catalog number 3516) and incubated for 24 hours at 37°C and 5% CO2. After incubation for 24 hours, cells were selected for two weeks with 2 ml complete growth medium containing 125 μg / mL hygromycin B (Thermo Fisher Scientific, catalog number 10687-010) and 225 μg / mL G418 (Thermo Fisher Scientific, catalog number 10131-027).

[0275] 9.3.1.4. Cell proliferation assay

[0276] Adherent cells (293T and HCT116) were separated by trypsin treatment. 1000-2000 cells / well were inoculated in 40 μL complete RPMI 1640 on 384-well tissue culture plates (Corning, catalog number 3764). Adherent cells were attached overnight. The next day, cells were treated with 10 μL of drugs at a specified concentration and incubated at 37°C with 5% CO2 for 5 days. At the end point, 40 μL of CellTiter-Glo luminescent cell viability assay reagent (Promega, catalog number G7573) was added to each well. Plates were read on a Molecular Devices SpectraMax M5 plate reader with a luminescent setting. Cell viability is expressed as a percentage relative to untreated controls.

[0277] 9.3.1.5. Immunofluorescence

[0278] RS4; 11 cell line was seeded on a V-bottom plate (Costar catalog number 3894) with 50k cells / well. Cells were Fc blocked with PBS+5% normal human serum (Sigma catalog number H4522) and 2% FBS. Wash cells and resuspend in staining buffer (PBS+2% FBS). Cells were stained with 5 μg / mL of anti-hu-CD19 antibody (CD19 mAb) conjugated with Alexa Flour 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 tracked at 37°C for 0.5 hours, 1 hour, 3 hours and 24 hours to induce internalization, and compared with cells stained on ice at T=0 time point control. Cells were then fixed and permeabilized (BD, catalog number 554714) using the manufacturing instructions. Cells were blocked with staining buffer containing 1% saponin + 2% mouse serum (Invitrogen catalog number 3881) and stained with 2 μg / mL Alexa Fluor 488-conjugated LAMP1 antibody (Biolegend, catalog number 328610) at 4°C overnight. Cells were washed 3 times in PBS containing 2% FBS. Cells were resuspended in imaging medium (FluroBrite: Gibco catalog number A1896701) and transferred to glass bottom imaging plates (Thermo Fisher Scientific, catalog number 160376) and analyzed on an IMX Micro confocal imager.

[0279] 9.3.1.6. Immunoblotting

[0280] 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 incubated in PBS supplemented with 1x Halt TMThe protein concentration of the lysate was measured by RIPA lysis buffer (Sigma-Aldrich, catalog number R0278) with a mixture of protease and phosphatase inhibitors (Thermo Fisher Scientific, catalog number 78446). The protein concentration of the lysate was measured with a BCA protein assay kit (Thermo Fisher Scientific, catalog number 23227). 10-20 μg of lysate was decomposed on a 4-12% gradient gel (Thermo Fisher Scientific, catalog number NW04120BOX) and transferred to a nitrocellulose membrane (ThermoFisher Scientific, catalog number IB23001). The membrane was incubated overnight at 4 ° C with a primary antibody at a concentration recommended by the manufacturer. The primary antibodies used in this study include phospho-AKT Ser473 (Cell Signaling Technology, catalog number 4060) or GAPDH (Cell Signaling Technology, catalog number 97166). The membrane was washed three times with 1×PBST (CellSignaling Technology, catalog number 9809) and then incubated with peroxidase AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, catalog number 115-035-003) or peroxidase AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, catalog number 111-035-003) for 1 hour at room temperature. The membrane was washed three times with 1×PBST and then incubated with Pierce TM The membrane was then incubated with ECL Western Blotting Substrate (Thermo Fisher Scientific, catalog number 32106) and detected using Azure Image Systems C600.

[0281] 9.3.1.7. Glucocorticoid response element (GRE) activation assay

[0282] K562 parental GRE (pGL4.36[luc2P / MMTV / Hygro]) and K562 human CD19 GRE (pGL4.36[luc2P / MMTV / Hygro]) or K562 cynomolgus CD19 GRE (pGL4.36[luc2P / MMTV / Hygro]) cells were seeded at 50,000 cells / well in 75 μL assay medium (1X RPMI+L-Glu, 1% CSFBS (Thermo Fisher Scientific Inc., Catalog No. 12676-029), 1% sodium pyruvate and 1% MEAA) on 96-well tissue culture treated white plates (Costar, Catalog No. 3917). GRE K562 parental cells or cells expressing human CD19 and cynomolgus CD19 were treated with 25 μL of 4× serially diluted CD19-GRM in assay medium and incubated for 72 hours at 37°C and 5% CO2. 100 nM dexamethasone was added as a positive control in these assays. After 72 hours of incubation, cells were treated with 100 μL Dual-Glo luciferase assay system (Promega catalog number E2920) for 10 minutes and analyzed for luminescence using MicroBeta (PerkinElmer). Dose response data were fitted for S-shaped curves using nonlinear regression, and EC50 values ​​were calculated using GraphPad 9.0 (GraphPad Software, Inc.).

[0283] 9.3.1.8.NFAT ADCC reporter gene assay

[0284] ADCC reporter gene bioassay (V and F variants; Promega, catalog number G7010 and G9790) was performed according to the manufacturer's protocol with slight modifications. Briefly, the target cell line Raji was seeded at 2500 cells / 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 specified antibody concentration. Effector cells from the V variant and F variant kits were resuspended in ADCC assay buffer and added at 5 μL per well to achieve an effector cell to target cell ratio of 6:1. The target cell-antibody mixture was incubated with effector cells expressing V variants and F variants at 37°C for 6 hours and 24 hours, respectively. At the endpoint, 15 μL of Bio-Glo luciferase reagent was added and luciferase activity was measured with a Molecular Devices SpectraMax M5 plate reader.

[0285] 9.3.1.9. PBMC Co-culture ADCC Assay

[0286] The target cell lines (RS4; 11, Raji and KARPAS422) were washed with PBS and labeled with 1 μM CFSE in PBS (Thermo Fisher Scientific, catalog number C34554) at 37°C for 5 minutes. The labeled target cells were then washed 3 times with medium containing 10% FBS. 20,000 labeled target cells in 50 μL were seeded onto a V-shaped 96-well plate. Antibodies and ADCs were titrated in the culture medium and 10 μL was added to reach the specified final concentration. PMBCs from a normal donor (AllCells) were added in 40 μL at a ratio of 20:1 effector cells to target cells, and the cells were incubated at 37°C for 4 hours. After incubation, the cells were washed once with PBS and stained with a live / dead fixable purple dead cell staining kit (Thermo Fisher Scientific catalog number L34955) at room temperature for 30 minutes, followed by washing once with 200 μL of ice-cold PBS. Cells were fixed with 4% paraformaldehyde in PBS for 15 min and then washed once with 200 μL PBS. Fixed cells were resuspended in 100 μL PBS and stored at 4 °C until analysis on a Stratedigm S1000EON flow cytometer. The percentage of live / dead purple dye-positive cells was captured and the percentage of specific lysis was calculated by subtracting the percentage of live / dead purple-positive cells for each treatment condition, where the untreated control contained only effector and target cells.

[0287] ADCP assay

[0288] Human monocytes (STEMCELL Technologies) were differentiated into macrophages in RPMI-1640 containing 10% FBS and 50ng / ml recombinant human M-CSF (BioLegend) for 8 days. In the phagocytosis assay, fully differentiated macrophages were separated from the flask for co-culture with target cancer cells. Human non-Hodgkin's lymphoma cell lines Raji and NuDHL1 expressing endogenous CD19 were selected as target cells. After washing with PBS, target cancer cells were labeled with 5μM CFSE dye solution (Thermo Fisher Scientific) for 15 minutes and then co-incubated with human macrophages at a ratio of 1:1. Different concentrations of CD19 mAb or CD19-GRM were added. After co-culture at 37°C for 3 hours, cells were harvested, washed and stained with antibodies against macrophage maturation markers, APC-Cy7-labeled CD68 antibodies (BioLegend), followed by flow cytometry analysis. The phagocytic index was calculated by counting the CFSE+CD68+ double positive macrophages among the total CD68+ macrophages, which shows the overall percentile of phagocytic macrophages.

[0289] 9.3.1.11. Apoptosis assay

[0290] Apoptosis was detected using Western blot assay. DLBCL cell lines Farage, SU-DHL-6, OCI-LY19 cells were treated with GRM payload (100 nM) or ADC-1 (1 μM) for the indicated time before cell lysis. TM Cell lysates were generated using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, catalog number 78501) and purified using Pierce TM Protein concentration was determined using a detergent compatibility assay kit (Thermo Fisher Scientific, catalog number 23246). Protein lysate (10 μg) from each treatment condition was added to a Bolt TM 4 to 12% Bis-Tris and iBlot TM 2 gel transfer apparatus onto a PVDF membrane. The membrane was blocked with 5% BSA and then immunoblotted with antibodies against the proteins of interest: BIM (Cell Signaling, catalog number 2933), caspase 3 (Cell Signaling, catalog number 9662), PARP (Cell Signaling, catalog number 9532), and GAPDH (Cell Signaling, catalog number 5174). The membrane was then probed with horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit or mouse IgG and imaged with SuperSignal on an Azure imager. TM The cells were detected using West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, catalog number 34580).

[0291] 9.3.2. In vivo studies

[0292] 9.3.2.1. Mice and husbandry

[0293] Female CB17 / SCID and SCID / beige were obtained from Charles River (Wilmington, MA), and CD34 +Humanized NSG IL-15 was obtained from Jackson Laboratory (Bar Harbor, ME) at 6 to 8 weeks of age and housed at a maximum of 10 per cage. Body weight upon arrival was 18 g to 20 g. Food and water were available ad libitum. Mice were acclimated to the animal facility for at least three days prior to the start of the experiments. Animals were tested during the light phase of a 12 h light: 12 h dark schedule (lights on at 06:00). All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of AbbVie and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

[0294] 9.3.2.2. Compounds

[0295] The intraperitoneal route of administration was used. GRM small molecules (GRM-SM) were formulated in an aqueous solution of 0.05% HPMC, 0.02% Tween-80 and stored at 4°C for oral administration.

[0296] The CD19 mAb from Example 1 is an afucosylated monoclonal antibody (IgG1, K) with high affinity to human and cynomolgus CD19. ADC-1 is a conjugate of the CD19 mAb with a bromoacetamide alanine-alanine peptide linker linked to a GRM of Formula V. The isotype antibody (AB095) recognizes a tetanus toxoid antigen that is neither displayed by xenografts nor by any murine tissue.

[0297] 9.3.2.3. Efficacy parameters

[0298] The magnitude of the therapeutic response parameters (maximum tumor growth inhibition, [TGI max ]) and persistence parameters (tumor growth delay, TGD) are used to indicate the efficacy of the drug. TGI indicates the difference between the mean tumor volume of the drug-treated group and the mean tumor volume of the 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. TGI max The value was determined when the difference between the treated and control groups was maximal. TGD (n) The indicated drug-treated groups reached a defined tumor volume (1 cm 3 The difference between the median time to reach the same volume in the vehicle-treated control group and the median time to reach the same volume in the vehicle-treated control group was expressed as a percentage of the median time to reach the specified tumor volume in the control group.

[0299] 9.3.2.4. Generation of tumor-bearing mice and determination of tumor volume of subcutaneous flank tumors

[0300] For each subcutaneous xenograft study, live cells were inoculated into the right flank of mice 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 sizes were matched to approximately 80 to 200 mm 3 Treatment was usually initiated within 24 hours after randomization and tumor size was matched to the desired cohort. At the start of treatment, mice weighed approximately 20-22 g. Tumor volume was estimated once to twice per week. Tumor length (L) and width (W) measurements were obtained via electronic calipers and volume was calculated according to the following formula: V = (L × W 2 ) / 2. When the tumor volume reaches 2,000mm 3 Euthanize mice according to institutional guidelines when the animal health is compromised or when the animal health is compromised.

[0301] Statistics

[0302] For TGI max Data from in vivo experiments were analyzed using Student's t-test and the log-rank test (Mann-Whitney U test or Mantel Cox) for TGD. Differences in response rates were assessed by Fisher's exact test.

[0303] 9.3.2.6.PDX studies

[0304] Patient-derived xenograft (PDX) studies were performed in a DLBCL model established in Wuxi, which was derived from surgically resected primary patient tumors. Patient treatment history was collected according to protocols and guidelines established by the Institutional Review Board. Each tumor sample (single cell suspension or tumor fragment) was prepared in Matrigel (BD Biosciences) and implanted into the flank of 6- to 8-week-old NOD scid mice. Mice were handled and housed according to IACUC / AALAS protocols and procedures. The size of the lesions (length × width) was measured twice weekly by caliper. 2 ) / 2 Measure the tumor when the tumor volume reaches approximately 150-200mm 3Treatment was started at 1:00. Mice were treated intraperitoneally with the test article once, as indicated by n=5 / group (vehicle, CD19 antibody, ADC-1). For each DLBCL PDX model tested (6 germinal center B cells (GCB) and 4 non-GCB, including a difficult-to-treat non-GCB activated B cell subtype (ABC)), tumor growth inhibition after ADC-1 treatment was determined as follows: Δ% TGI max = 1-(tumor volume of the treatment group on day X-tumor volume of the treatment group at randomization) / (tumor volume of the control on day X-tumor volume of the control at randomization)]*100. Δ% TGI max was determined when the difference between the treatment group and the control group was the largest. PDX models were determined as responders or non-responders based on their ability to induce tumor regression: (Responders): >105% ΔTGI max-yes / no response. Tumor volume change (%) was also calculated and was calculated as [(tumor volume at time point / tumor volume at randomization)-1]×100, based on the percentage of tumors reaching 1000 mm in vehicle control groups. 3 Graph the time points. Figure 13A-13B .

[0305] 9.3.27. Binding activity

[0306] ADC-1 was evaluated for cell surface binding to different cancer cell lines endogenously expressing CD19 and 293T cells transduced with full-length human CD19 using flow cytometry. ADC-1 exhibited binding EC ranging from 0.544 nM to 1.061 nM on 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 displayed a binding EC of 1.086 nM on 293T cells engineered with full-length human CD19. 50 (Table 4) The unconjugated parental afucosylated CD19 antibody (CD19 mAb) of Example 1 exhibited similar binding affinity on 293T cells engineered with full-length human CD19, EC 50 The range was 0.401 nM to 0.836 nM (Table 4), with EC 50 The concentration of ADC-1 and CD19 mAb was 0.977 nM (Table 4). Neither ADC-1 nor CD19 mAb showed detectable binding to the CD19 negative colorectal cancer cell line HCT116.

[0307] Table 4. Binding affinity of ADC-1 to cell CD19

[0308]

[0309] ALL = acute lymphoblastic leukemia; BL = Burkitt's lymphoma; CRC = colorectal cancer; DLBCL = diffuse large B-cell lymphoma, nd = not detected

[0310] a. Shows the average EC from two independent experiments 50 .

[0311] 9.3.2.8. Glucocorticoid Receptor Modulator (GRM) Payloads More Effective Than Clinically Approved Glucocorticoids

[0312] The potency of the GRM payload was compared to clinically approved glucocorticoids (dexamethasone and prednisolone) by testing RS4;11 in vitro. The GRM payload of Formula IV showed higher potency ( Figure 1 ). The GRM payload of Formula IV, the EC of dexamethasone and prednisolone 50 They are 0.23nM, 3.28nM and 82.91nM respectively.

[0313] 9.3.2.9. CD19 mAb and ADC-1 Internalize and Deliver GRM Payload to Inhibit B Cell Lymphoma Cell Proliferation

[0314] To investigate the ability of CD19 mAb to induce CD19 internalization, the acute lymphoblastic lymphoma (ALL) cell line RS4;11 was treated with CD19 mAb at 37°C for 0, 0.5, 1, 3, and 24 hours. CD19 mAb induced CD19 internalization and localization to lysosomes as early as 0.5 hours, as indicated by the colocalization of CD19 with the lysosomal marker LAMP1 ( Figure 2 ). Similarly, ADC-1 activated the GRE reporter in a dose-dependent manner in K562 glucocorticoid response element (GRE) reporter cells engineered to express human CD19 ( Figure 3 ).

[0315] 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 cancer cell lines expressing CD19 that were sensitive to the glucocorticoid receptor modulator (GRM) payload of Formula IV and the in vitro cytotoxicity EC of ADC-1 was 50 The range was 0.082nM-0.321nM (Table 5).

[0316] Table 5. CD19 expression in in vitro cell lines and sensitivity to treatment with ADC-1 and Formula IV

[0317]

[0318] nd = Not Detected

[0319] DLBCL = diffuse large B-cell lymphoma, BL = Burkitt's lymphoma, ALL = acute lymphoblastic lymphoma, CRC = colorectal cancer

[0320] a. Approximate number of cell surface CD19 molecules per cell as determined by flow cytometry, as antibody binding capacity for CD19 mAb binding at 67 nM.

[0321] b. Shows the average EC of the antiproliferation assay from two independent experiments. 50 .EC 50 Values ​​represent complete cell killing as observed for Formula IV and CD19-GRM.

[0322] The GRM prodrug payload of formula III is dephosphorylated in vitro / in vivo to the dephosphorylated GRM payload of formula IV, so the dephosphorylated payload of formula IV is used as a control in our study. ADC-1 has no activity on the CD19 negative colorectal cancer cell line HCT116 (Table 5). It is worth noting that ADC-1 is more effective than isotype-GRM (Table 5), indicating the CD19 target-dependent delivery of GRM payload.

[0323] 9.3.2.10. CD19 mAb inhibits DLBCL cell proliferation and AKT activation in vitro

[0324] CD19 is a co-receptor of the B cell receptor and can transactivate the PI3K / AKT pathway (Burger et al., Nat Rev Cancer, 2018.18(3): pp. 148-167). In addition, functional genomic screening demonstrated that the survival of a subset of DLBCL cell lines depends on CD19 (Phelan, JD et al., Nature, 2018.560(7718): pp. 387-391). CD19 mAb (from Embodiment 1) treatment inhibited cell proliferation of SU-DHL-6 in vitro ( Figure 4A Pretreatment with CD19 mAb abolished AKT phosphorylation in response to BCR stimulation with 1 μg / ml anti-IgM ( Figure 4B ), demonstrating that CD19 mAb can effectively block BCR-mediated AKT pathway activation.

[0325] 9.3.2.11. ADC-1 mediates antibody-dependent cellular cytotoxicity (ADCC)

[0326] CD19 mAb and ADC-1 have been afucosylated to enhance their ability to elicit antibody-dependent cellular cytotoxicity (ADCC). Polymorphisms on FcγPIIIa (F158 or V158) affect its binding to IgG1, IgG3 and IgG4 and the therapeutic efficacy of these antibodies (Koene, HR et al., Blood, 1997. 90(3): pp. 1109-1114; Cartron, G. et al., Blood, 2002. 99(3): pp. 754-758; Wu, J. et al., J Clin Invest, 1997. 100(5): pp. 1059-1070).

[0327] NFAT reporter Jurkat cells engineered to express high affinity (V158) or low affinity (F158) FcγRIIIa were used to determine the ability of CD19-GRM to bind FcγRIIIa and elicit ADCC in vitro. CD19-GRM was expressed with EC values ​​of 0.017 nM and 0.018 nM on the V158 and F158 variants, respectively. 50 Induces ADCC NFAT reporter activation ( Figure 5A and Figure 5B Similarly, the unconjugated afucosylated antibody CD19 mAb also bound to the V158 and F158 variants with EC values ​​of 0.027 nM and 0.023 nM, respectively. 50 Induces potent ADCC activity ( Figure 5A and Figure 5B ).

[0328] Co-culture of B cell lymphoma cell lines with primary PBMCs was performed to functionally evaluate the ADCC activity of ADC-1. In these assays, ADC-1 elicited CD-19-specific cancer cell lysis, EC 50 The range is 0.07nM-0.142nM ( Figure 6A-6C Unconjugated CD19 mAb also demonstrated similar CD19-specific cancer cell lysis, EC 50 The range is 0.075nM-0.405nM ( Figure 6A-6C ).

[0329] 9.3.2.12. ADC-1 induces antibody-dependent cellular phagocytosis (ADCP)

[0330] The ability of ADC-1 to induce ADCP was evaluated by co-culture of B cell lymphoma cells with monocyte-derived macrophages in vitro. In NuDHL1 and Raji, both ADC-1 and CD19 mAb treatment resulted in significantly higher ADCP induction compared with isotype mAb or isotype-GRM treatment ( Fig. 7A and Figure 7B ).

[0331] 9.3.2.13. GRM and ADC-1 induce apoptosis in DLBCL cell lines

[0332] GRM and ADC1 induced apoptosis as measured by upregulation of a pro-apoptotic protein (BIM) and induction of apoptotic markers (cleaved caspase-3 and cleaved PARP) in all three cell lines tested (see Fig.15 ).

[0333] 9.3.2.14. ADC-1 induces GRM-driven antitumor activity and ADCC in vivo

[0334] ADC-1 is effective in tumors expressing CD19 on the cell surface. 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 Fig. 9 Immunocompromised CB17 / SCID mice (OCI-LY19, RS4; 11: Fig. 8A and Fig. 9 ), immunocompromised SCID / beige mice (SU-DHL-6: Figure 8B ) and humanized CD34+NSG-huIL-15 mouse models (OCI-LY19: Figure 10) were used to study human xenografts. ADC-1 was effective in a dose-dependent manner as a single bolus intraperitoneally. In DLBCL xenografts (Figure 8), all doses of ADC-1 and glucocorticoid receptor modulator small molecule (GRM-SM) of Formula IV induced statistically significant (p < 0.05) tumor growth inhibition, which was greater than vehicle, isotype monoclonal antibodies, human CD19 monoclonal antibodies, and systemic steroids (prednisolone, daily administration). Significant durable responses were observed at the highest dose in DLBCL xenografts. AB095-GRM (isotype-GRM control) showed some growth inhibition at the two highest doses, which was significantly lower (p < 0.05) than ADC-1 at the same dose. ADC-1 ( Fig. 9 ) showed high sensitivity, with durable responses at low doses, and statistically significant (p<0.05) tumor growth inhibition. Tumor regressions at dose 2 lasted for >100 days. All doses were tolerated and no weight loss was observed.

[0335] ADC-1 is effective in a dose-dependent manner in an antibody-dependent cellular cytotoxicity (ADCC)-compatible mouse model (huCD34+NSG-huIL-15). In this mouse model, immunocompromised NSG mice are genetically engineered to express human IL-15 and implanted with human PBMCs (CD34+). Human IL-15 supports NK cell viability and function, enabling ADCC studies. In this mouse system, ADC-1 induces inhibition of subcutaneous xenograft growth 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 day, 6 days, 13 days, 20 days, and 26 days after dosing. For all ADC-1 dose levels, the number of human B cells per microliter of mouse blood and the percentage of total human CD45+ cells were reduced. The duration of B cell suppression was positively correlated with dose level. NK cell numbers were not significantly affected by ADC-1 at any dose level.

[0336] The efficacy of ADC-1 was also directly compared to the combination of CD19 mAb and systemic prednisolone in the RS4;11 xenograft model. In this model, high doses of single agent CD19 mAb or prednisolone (QDx5]x3) demonstrated modest antitumor activity, while the combination of the two agents resulted in more sustained tumor growth inhibition ( Fig.11 Notably, treatment with a single low dose of ADC-1 induced more durable antitumor activity compared with the combination of CD19 mAb and systemic prednisolone ([QDx5]x3).

[0337] ADC-1 has been shown to be effective in many different B cell malignancy models DB ( Fig. 12A )、SUPB15( Fig. 12B )、RS4;11( Fig. 12C ) demonstrated antitumor efficacy in ALL tumor models. As shown, the antitumor efficacy of ADC-1 was directly compared with prednisolone [QDx5]x3 or isotype-GRM ADC in DB and SUPB15 xenograft models grown in immunocompromised mice. Sustained antitumor efficacy was observed in both DB and SUPB15 xenograft models, outperforming systemic steroid prednisolone and outperforming isotype-GRM ADC, confirming the CD19-targeting activity of ADC-1. ALL tumor model SUPB15 ( Fig. 12B ) demonstrated sustained antitumor activity (complete response) lasting more than 50 days after a single ADC-1 dose. RS4;11 tumors ( Fig. 12C ) grow to large size (>600mm 3The patients were then treated with a single IP dose of ADC-1. This single ADC-1 administration caused complete tumor regression of these large tumors, which lasted for >40 days.

[0338] 9.3.2.15. ADC-1 induces anti-tumor activity in DLBCL PDX models

[0339] like Fig.13A As shown, all 10 DLBCL PDX models exhibited tumor growth inhibition relative to control-treated PDX of the same model. Using a single dose of ADC-1 at dose 3, 9 out of 10 models showed tumor regression (see Fig. 13B ). Anti-tumor efficacy was seen in PDX models derived from GCB and non-GCB patients. Only one model, 0232, did not show stagnation or regression when treated with ADC-1 at dose 3. This PDSX model is from a patient who has undergone 7 cycles of R-CHOP treatment. Three PDX models (0367, 0016, and 0207) from patients with R-CHOP relapse who had been treated for 4 cycles did respond.

[0340] like Fig.14A and Fig. 14B As shown in , a single dose of ADC-1 at dose 3 exhibited anti-tumor activity when compared to vehicle control and CD19 antibody treatment. Fig.14A In this study, a PDX model was derived from a DLBCL, GCB patient who had been treated with 4 cycles of R-CHOP. This PDX model showed mild stagnation when treated with ibrutinib. Fig. 14B The PDX-derived model shown in is from a DLBCL ABC patient who had received 4 cycles of R-CHOP. This PDX model was resistant to ibrutinib treatment.

Claims

1. An anti-CD19 antibody-drug conjugate, comprising the following structure: in: A is antibody; R is the point of attachment of the antibody formed through a cysteine ​​residue of the antibody, which upon attachment 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; Wherein the antibody is an anti-CD19 antibody, and the anti-CD19 antibody comprises 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, wherein 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).

2. The anti-CD19 antibody-drug conjugate according to claim 1, wherein the antibody is an IgG antibody.

3. The anti-CD19 antibody-drug conjugate according to claim 2, wherein the antibody is an IgG1 antibody.

4. The anti-CD19 antibody-drug conjugate of claim 1, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

8.

5. The anti-CD19 antibody-drug conjugate of claim 1, wherein the antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

10.

6. The anti-CD19 antibody-drug conjugate according to any one of claims 1 to 5, wherein the antibody is afucosylated.

7. The anti-CD19 antibody-drug conjugate according to claim 3 or 4, wherein R 1 It is -P(=O)(OH)2.

8. The anti-CD19 antibody-drug conjugate according to claim 3 or 4, wherein R 1 Yes -H.

9. The anti-CD19 antibody-drug conjugate according to any one of claims 6 to 8, wherein the afucosylation is at position ASN-300 (EU: ASN-297).

10. A pharmaceutical composition comprising a population of anti-CD19 antibody-drug conjugates according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the antibody is about 70% or more, 80% or more, 90% or more, or about 99%, or about 100% afucosylated.

12. A method of treating a B-cell malignancy, the method comprising administering to a patient in need thereof an antibody-drug conjugate according to any one of claims 1 to 9.

13. 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, wherein 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).

14. The anti-CD19 antibody according to claim 13, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

8.

15. The anti-CD19 antibody according to claim 13, wherein the antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

10.

16. The anti-CD19 antibody according to any one of claims 13 to 15, wherein the antibody is afucosylated.

17. The anti-CD19 antibody of claim 16, wherein the afucosylation is at position ASN-300 (EU: ASN-297).

18. An anti-CD19 antibody-drug conjugate, comprising the following structure: in: A is antibody; R is the point of attachment of the antibody formed through a cysteine ​​residue of the antibody, which upon attachment 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; The antibody is an anti-CD19 antibody, and the anti-CD19 antibody comprises two heavy chains and two light chains, each heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, and each light chain consisting of the amino acid sequence shown in SEQ ID NO: 10; and wherein the antibody is afucosylated at position ASN-303 (EU: ASN-297).

19. The anti-CD19 antibody of claim 18, wherein n is 2, 4, 6 or 8.

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