JPWO2020092654A5 - - Google Patents

Description

[Cross-reference of related applications]
This application is filed on October 30, 2018, US Provisional Application No. 62 / 753,002, November 30, 2018, US Provisional Application No. 62 / 774,006, and February 19, 2019. Claims priority over the filed US Provisional Application No. 62 / 807,582. The content of each preferred application is incorporated into this application by reference.

[Technical field]
The present invention relates to the field of anti-CD45 antibody or antibody drug conjugate thereof. The present invention further relates to the treatment of patients suffering from various pathological conditions such as blood disorders, metabolic disorders, cancer, and autoimmune diseases by administration of anti-CD45 antibody or antibody drug conjugate (ADC). Here, the antibody or ADC can bind to CD45 on the surface of either hematopoietic stem cells or immune cells.

CD45 is a member of the protein tyrosine phosphatase (PTP) family and is a 180-240 kD transmembrane glycoprotein. CD45 is a signal transduction molecule that regulates many cellular processes such as cell proliferation and differentiation. CD45 is a type I transmembrane protein found as a variety of isoforms on the surface of differentiated hematopoietic cells, and has an important role in T-cell and B-cell receptor signaling. Has been shown. In general, except for mature erythrocytes and platelets, cells of hematopoietic origin express CD45.

Different isoforms of the CD45 exist because the exon undergoes a variety of splicings. Various isoforms of human CD45 differ in their extracellular domains and are expressed differently on subpopulations of B- and T-cell lymphocytes. At least six different human CD45 mRNA isoforms have been isolated, including all three exons (ABC isoforms), two of the three exons (AB and BC isoforms), and one exon. Only (A isoform and B isoform), or exon-free (O isoform) (Hermiston et al. (2003) Annu Rev Immunol. 21, 107-137.). The suffix RA, RB, or RO requires an amino acid residue corresponding to exon A (RA), exon B (RB), or exon A, B, and C for CD45 antigen expression. It shows the lack of a corresponding amino acid residue (RO).

Despite advances in medical technology, there is still a need to treat the pathophysiology of the hematopoietic system, especially certain blood cell disorders, metabolic disorders, cancer, and autoimmune symptoms. While hematopoietic stem cells (HSCs) have great therapeutic potential, they have limitations that impede clinical use due to the difficulty in reliably engrafting HSC grafts in the host. In particular, hematopoietic stem cell therapy, which includes antibodies targeting cell surface antigens on endogenous HSCs, induces unwanted immunostimulatory and effector functions that prevent exogenous HSC transplants from engrafting. There is. Needs for targeted therapies that affect both HSC and immune cells to improve transplantation (eg, CD45-targeted therapies as antigens capable of conditioning patients for HSC transplantation) However, it still exists in the art.

In this application, anti-CD45 antibodies (anti-human CD45 9 (hCD45) antibodies) and antibody drug conjugates (ADCs) that bind to hematopoietic stem cells (HSCs) and are useful, for example, as conditioning agents for transplantation. ) Is described. In particular, the anti-CD45 antibodies and ADCs described in this application can be used to specifically reduce host HSCs, immune cells and disease-causing cells. In addition, the anti-CD45 antibodies and ADCs described in this application are used to treat patients with leukemia or lymphoma, as well as patients with autoimmune diseases such as multiple sclerosis and scleroderma. Sometimes. The anti-CD45 antibodies and ADCs described in this application ensure that engraftment of exogenous hematopoietic stem cell transplants is preserved after transplantation with the multi-potency and hematopoietic functionality of these cells. The needs of compositions and methods for facilitating are met.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is described in CDR1, SEQ ID NO: 3 having the amino acid sequence set forth in SEQ ID NO: 2. Contains a heavy chain comprising a variable region comprising CDR2 having the amino acid sequence of, CDR3 having the amino acid sequence set forth in SEQ ID NO: 4, and the amino acid set forth in SEQ ID NO: 6. A light chain comprising a variable region comprising CDR1 having a sequence, CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 8. include.

In certain embodiments, the isolated anti-CD45 antibody, or antigen binding moiety thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or SEQ ID NO: 1. (SEQ ID NO): Contains a light chain variable region comprising the amino acid sequence set forth in 5.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is described in CDR1, SEQ ID NO: 11 with the amino acid sequence set forth in SEQ ID NO: 10. Contains a heavy chain comprising a variable region comprising CDR2 having the amino acid sequence of, CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and the amino acid set forth in SEQ ID NO: 14. A light chain comprising a variable region comprising CDR1 having a sequence, CDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 16. include.

In certain embodiments, the isolated anti-CD45 antibody, or antigen binding moiety thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and / or SEQ ID NO: (SEQ ID NO): Contains a light chain variable region comprising the amino acid sequence set forth in 13.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is described in CDR1, SEQ ID NO: 19 having the amino acid sequence set forth in SEQ ID NO: 18. Contains a heavy chain comprising a variable region comprising CDR2 having the amino acid sequence of, CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and the amino acid set forth in SEQ ID NO: 22. A light chain comprising a variable region comprising CDR1 having a sequence, CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, CDR3 having the amino acid sequence set forth in SEQ ID NO: 24. include.

In certain embodiments, the isolated anti-CD45 antibody, or antigen binding moiety thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and / or SEQ ID NO: (SEQ ID NO): Contains a light chain variable region comprising the amino acid sequence set forth in 21.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is set forth in CDR1, SEQ ID NO: 3 having the amino acid sequence set forth in SEQ ID NO: 2. Contains a heavy chain comprising a variable region comprising CDR2 having the amino acid sequence of, CDR3 having the amino acid sequence set forth in SEQ ID NO: 4, and the amino acid set forth in SEQ ID NO: 6. A light chain comprising a variable region comprising CDR1 having a sequence, CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 8. Containing, isolated anti-CD45 antibody, or antigen-binding portion thereof; comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or SEQ ID NO. ): An isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a light chain variable region comprising the amino acid sequence set forth in 5; SEQ ID NO: 10; CDR1, having the amino acid sequence set forth in 10. Contains a heavy chain comprising a variable region comprising CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and SEQ ID NO: (SEQ ID NO): CDR1 having the amino acid sequence set forth in 14, SEQ ID NO: CDR2 having the amino acid sequence set forth in 15, SEQ ID NO: 16 having the amino acid sequence set forth in 16. An isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a light chain comprising a variable region comprising CDR3,; comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9. , And / or an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13; or SEQ ID ID. NO): CDR1 having the amino acid sequence set forth in 18; CDR2 having the amino acid sequence set forth in SEQ ID NO: 19; CDR3 having the amino acid sequence set forth in SEQ ID NO: 20. Contains heavy chains containing variable regions containing, and SEQ ID NO: 22. Contains a variable region comprising CDR1 having the amino acid sequence set forth in the above, CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, CDR3 having the amino acid sequence set forth in SEQ ID NO: 24. Binds to the same epitope as the isolated anti-CD45 antibody, or antigen-binding portion thereof, including the light chain.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 containing residues 486R, 493Y, and 502T of SEQ ID NO: 37. And binds to cynomolgus monkey and lizard monkey CD45; or specifically binds to an epitope of human CD45 containing the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38), and binds to cypress monkey and lizard monkey CD45; or , Specificly binds to an epitope of human CD45 containing the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 39), and binds to cynomolgus monkey and red-tailed monkey CD45.

In another embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is specific for an epitope of human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 37. Binds; in peptides containing RNGPHERYHLEVEAGNT (SEQ ID NO: 38), at least one additional amino acid, at least two additional amino acids, at least three additional amino acids, at least four additional amino acids, or Binds to at least 5 additional amino acids, where the residues of the additional amino acids are not residues 486R, 493Y, and 502T of SEQ ID NO: 37; and / or cynomolgus monkeys and Binds to red-throated monkey CD45.

In another embodiment, anti-CD45 specifically binds to an epitope of human CD45 containing residues 486R, 493Y, and 502T of SEQ ID NO: 37, and binds to cynomolgus monkey and rhesus monkey CD45. Antibodies, or antigen-binding moieties thereof, are provided in this application.

In yet another embodiment, an anti-CD45 antibody, or anti-CD45 antibody, that specifically binds to an epitope of human CD45 comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38) and binds to cynomolgus monkey and rhesus monkey CD45. The antigen binding moiety is provided in this application.

In a further embodiment, an anti-CD45 antibody, or an anti-CD45 antibody thereof, that specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 39) and binds to cynomolgus monkey and rhesus monkey CD45. Antigen binding moieties are provided in this application.

In certain embodiments, the antibody competes with antibody AbA for binding to human CD45.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is the de-immunized antibody described in this application.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is a chimeric antibody (eg, chimeric AbA, AbB, or AbC).

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is a humanized antibody (eg, humanized AbA, AbB, or AbC).

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is an intact antibody (eg, an intact IgG antibody).

In another embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to human CD45 and crab monkey CD45, an isolated deimmunized monoclonal anti-CD45 antibody, or antigen thereof. The anti-CD45 antibody according to any one of claims 1 to 15, which is a binding portion, or an antigen-binding portion thereof is a parent antibody of the deimmunized anti-CD45 antibody or an antigen-binding portion thereof. ..

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, has at least one amino acid substitution of H435 or I235 / H310 / H435 (EU numbered) (eg, H435A (EU numbered), I235A. Includes Fc region including being / H310A / H435A (EU numbering)).

In certain embodiments, the isolated anti-CD45 antibody, or antigen binding site thereof, is IgG (eg, IgG1 or IgG4).

In certain embodiments, a feature of the invention is an antibody drug conjugate (ADC) comprising an anti-CD45 antibody disclosed in this application that is bound to a cytotoxin via a linker.

In some embodiments, the cellular toxins are amatoxin, pseudomonas exotoxin A, de bouganine, diphtheria toxin, saporin, maytancin, maytancinoid, auristatin, anthraciclin, calikeamycin, irinotecan, SN-38, duo. Carmycin, pyrolobenzodiazepine, pyrolobenzodiazepine dimer, indolinobenzodiazepine, or indolinobenzodiazepine dimer.

In some embodiments, the cytotoxin is a microtubule binder or RNA polymerase inhibitor. In certain embodiments, the cytotoxin is an RNA polymerase inhibitor.

In certain embodiments, the RNA polymerase inhibitor is amatoxin.

In some embodiments, the amatoxin is α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, or proamanullin.

ここで、R₁は、H、OH、OR_A、又はOR_Cである；
R₂は、H、OH、OR_B、又はOR_Cである；
R_A 及びR_Bは、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、R_C、又はR_Dである；
R₄、R₅、R_6, 及びR₇は、それぞれ独立してH、OH、OR_C、OR_D、R_C、又はR_Dである；
R₈は、OH、NH₂、OR_C、OR_D、NHR_C、又はNR_CR_Dである；
R₉は、H、OH、OR_C、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；
R_Cは、-L-Zである；
R_Dは、任意選択的に置換されたC₁-C₆アルキル、任意選択的に置換されたC₁-C₆ヘテロアルキル、任意選択的に置換されたC₂-C₆アルケニル、任意選択的に置換されたC₂-C₆ヘテロアルケニル、任意選択的に置換されたC₂-C₆アルキニル、任意選択的に置換されたC₂-C₆ヘテロアルキニル、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである；
Lは、任意選択的に置換されたC₁-C₆アルキレン、任意選択的に置換されたC₁-C₆ヘテロアルキレン、任意選択的に置換されたC₂-C₆アルケニレン、任意選択的に置換されたC₂-C₆ヘテロアルケニレン、任意選択的に置換されたC₂-C₆アルキニレン、任意選択的に置換されたC₂-C₆ヘテロアルキニレン、任意選択的に置換されたシクロアルキレン、任意選択的に置換されたヘテロシクロアルキレン、任意選択的に置換されたアリーレン、任意選択的に置換されたヘテロアリーレン、ペプチド、ジペプチド、-(C=O)-、又はそれらの組合せである；並びに、
Zは、L上に存在する反応性置換基と、前記抗体又は抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される化学的な部分構造である。 In certain embodiments, the amatoxin is represented by formula (IV).

Where R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
_RA and R _B , together with the oxygen atom to which they are attached, form an arbitrarily substituted 5-membered heterocycloalkyl group;
R ₃ is H, R _C , or R _D ;
R ₄ , R ₅ , R _6, and R ₇ are independently H, OH, OR _C , OR _D , _RC , or R _D ;
R ₈ is OH, NH ₂ , OR _C , OR _D , NHR _C , or NR _C R _D ;
R ₉ is H, OH, OR _C , or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
_RC is -LZ;
R _D is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted. C ₂ -C ₆ heteroalkenyl substituted with, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted C ₂ -C ₆ heteroalkynyl, optionally substituted cycloalkyl , Optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C ₁ -C ₆ alkylene, optionally substituted C ₁ -C ₆ heteroalkylene, optionally substituted C ₂ -C ₆ alkenylene, optionally substituted. Substituted C ₂ -C ₆ heteroalkenylene, optionally substituted C ₂ -C ₆ alkynylene, optionally substituted C ₂ -C ₆ heteroalkynylene, optionally substituted cycloalkylene , Optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, peptide, dipeptide,-(C = O)-, or a combination thereof; and,
Z is a chemical partial structure formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within said antibody or antigen binding fragment.

ここで、
R₁は、H、OH、OR_A、又はOR_Cである；
R₂は、H、OH、OR_B、又はOR_Cである；
R_A 及びR_Bは、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、R_C、又はR_Dである；
R₄、R₅、R_6, 及びR₇は、それぞれ独立してH、OH、OR_C、OR_D、R_C、又はR_Dである；
R₈は、OH、NH₂、OR_C、OR_D、NHR_C、又はNR_CR_Dである；
R₉は、H、OH、OR_C、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；
R_Cは、-L-Zである；
R_Dは、任意選択的に置換されたC₁-C₆アルキル、任意選択的に置換されたC₁-C₆ヘテロアルキル、任意選択的に置換されたC₂-C₆アルケニル、任意選択的に置換されたC₂-C₆ヘテロアルケニル、任意選択的に置換されたC₂-C₆アルキニル、任意選択的に置換されたC₂-C₆ヘテロアルキニル、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである；
Lは、任意選択的に置換されたC₁-C₆アルキレン、任意選択的に置換されたC₁-C₆ヘテロアルキレン、任意選択的に置換されたC₂-C₆アルケニレン、任意選択的に置換されたC₂-C₆ヘテロアルケニレン、任意選択的に置換されたC₂-C₆アルキニレン、任意選択的に置換されたC₂-C₆ヘテロアルキニレン、任意選択的に置換されたシクロアルキレン、任意選択的に置換されたヘテロシクロアルキレン、任意選択的に置換されたアリーレン、若しくは任意選択的に置換されたヘテロアリーレン、ペプチド、ジペプチド、-(C=O)-、又はそれらの組合せである；並びに、
Zは、L上に存在する反応性置換基Z'と、前記抗体又は抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される化学的な部分構造である、
ここで、Amは正確に1つのR_C置換基を含む。 In one embodiment, the amatoxin-linker conjugate (Am-LZ) is represented by the formula (IA).

here,
R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
_RA and R _B , together with the oxygen atom to which they are attached, form an arbitrarily substituted 5-membered heterocycloalkyl group;
R ₃ is H, R _C , or R _D ;
R ₄ , R ₅ , R _6, and R ₇ are independently H, OH, OR _C , OR _D , _RC , or R _D ;
R ₈ is OH, NH ₂ , OR _C , OR _D , NHR _C , or NR _C R _D ;
R ₉ is H, OH, OR _C , or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
_RC is -LZ;
R _D is optionally substituted C ₁ -C ₆ alkyl, optionally substituted C ₁ -C ₆ heteroalkyl, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted. C ₂ -C ₆ heteroalkenyl substituted with, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted C ₂ -C ₆ heteroalkynyl, optionally substituted cycloalkyl , Optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C ₁ -C ₆ alkylene, optionally substituted C ₁ -C ₆ heteroalkylene, optionally substituted C ₂ -C ₆ alkenylene, optionally substituted. Substituted C ₂ -C ₆ heteroalkenylene, optionally substituted C ₂ -C ₆ alkynylene, optionally substituted C ₂ -C ₆ heteroalkynylene, optionally substituted cycloalkylene , Optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene, peptide, dipeptide,-(C = O)-, or a combination thereof. ;and,
Z is a chemical partial structure formed from the coupling reaction between the reactive substituent Z'present on L and the reactive substituent present within said antibody or antigen binding fragment.
Here, Am contains exactly one _RC substituent.

である。 In some embodiments, the LZ

Is.

ここで、Abは、前記抗-CD45抗体に結合する位置を表す。 In certain embodiments, the ADC has the formula Am-LZ-Ab and is represented by:

Here, Ab represents a position that binds to the anti-CD45 antibody.

である。 In some embodiments, the linker-chemical partial structure LZ,

Is.

である。 In some embodiments, the LZ

Is.

ここで、Abは、抗-CD45抗体に結合する位置を表す。 In certain embodiments, the ADC has the formula Am-LZ-Ab and is represented by:

Here, Ab represents a position that binds to the anti-CD45 antibody.

In certain embodiments, the RNA polymerase inhibitor is amanitin.

In certain embodiments, the cellular toxins are pseudomonas exotoxin A, de bouganine, diphtheria toxin, saporin, maytancin, maytancinoid, auristatin, anthraciclin, calikeamycin, irinotecan, SN-38, duocarmycin, pyrrolo. It is selected from the group consisting of benzodiazepines, pyrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers, or indolinobenzodiazepine pseudodimers.

In some embodiments, the cytotoxin is a microtubule binder or auristatin. In some embodiments, the microtubule binder is maitansine. In some embodiments, the microtubule binder is a maytansinoid. In some embodiments, the mayantinoid is selected from the group consisting of DM1, DM3, and DM4, as well as maytancinol.

In certain embodiments, the auristatin is MMAE or MMAF.

In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

In certain embodiments, the antibody is conjugated to the toxin by a cysteine residue in the Fc domain of the antibody.

In certain embodiments, the cysteine residue is introduced by amino acid substitution in the Fc domain of the antibody.

In certain embodiments, the amino acid substitution is D265C and / or V205C (EU numbering).

In certain embodiments, the ADC has a drug-to-antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7, or 8.

Also provided are pharmaceutical compositions comprising the antibodies or ADCs described in this application and pharmaceutically acceptable carriers.

Further provided is a method of reducing a population of hematopoietic stem cells (HSCs) in a human patient, wherein the method comprises administering to the patient an effective amount of an antibody or ADC according to the present application. In certain embodiments, the method further comprises administering to the patient a graft comprising hematopoietic stem cells.

In certain embodiments, the graft is allogeneic.

In certain embodiments, the implant is self.

Also provided is a method comprising administering to a human patient a transplant containing hematopoietic stem cells, wherein the patient is in an amount sufficient to reduce the population of hematopoietic stem cells in the patient. The antibody or ADC described in 1 has been administered in advance.

In certain embodiments, the hematopoietic stem cell is a CD45 + cell.

In certain embodiments, the patient has a blood disorder, metabolic disorder, cancer, or autoimmune disease, or severe combined immunodeficiency disease (SCID).

Also disclosed are methods of treating leukemia in human patients, wherein said method comprises administering to a human patient having leukemia the antibody or ADC described in this application.

Further provided, a method comprising administering to a human patient a transplant containing hematopoietic stem cells, wherein the patient is in an amount sufficient to reduce the population of immune cells in the patient, the present application. The antibody or ADC described in 1 has been administered in advance.

In certain embodiments, the immune cell is a CD137 +, CD2 +, or CD5 + cell.

In certain embodiments, the immune cell is a T cell.

FIGS. 1A, 1B, 1C, 1D and 1E show that anti-CD45 amanitin antibody drug conjugate (CD45-Am) enables in vitro and in vivo reduction of lymphocytes and hematopoietic stem cells (HSC). The results of the experiment are shown in a graph. 2A and 2B graphically show the results of an in vitro cell injury assay showing that anti-CD45-AM is very effective in injuring human HSCs in vitro. In FIG. 2A, the human PBMC is represented by the presence of an anti-CD45-amanitine conjugate (antibody AbB-amanitin; "CD45-AM") or a control non-targeting isotype compatible-ADC ("isotype-AM"). They were cultured under 4 days and cell viability was measured by luminescence by Celltiter Glo (x-axis) with varying antibody concentrations (RLU; y-axis). In FIG. 2B, primary human CD34 ⁺ bone marrow cells are cultured in CD45-AM or isotype-AM for 5 days, and surviving CD34 + CD90 + HSCs are flow cytometry with varying antibody concentrations (x-axis). Counted by (y-axis). FIGS. 3A and 3B are xenograft mouse models inoculated with human leukemia cells derived from an immortalized cell line expressing CD45 (a luciferase-labeled ALL cell line expressing CD45 (5x10 ⁶ cells / mouse)). The results of the in vivo survival test using the above are shown in a graph. These indicate that anti-CD45-AM (AbB bound to amanitin) doubles median survival up to 40 days in the REH-Luc xenograft model. FIG. 3A shows a single injection of 1 mg / kg anti-CD45-AM on day 5 after inoculation with ALL compared to controlled or unconjugated anti-CD45 antibody treated with vehicle (PBS). It is shown that the median survival was longer than 15 days (n = 10 mice / group, p <0.0001). FIG. 3B shows typical pseudocolor images of bioluminescent signals obtained using the IVIS Imaging System (Perkin Elmer) 19 days post-implantation for the anti-CD45-Am and isotype-AM treatment groups. show. FIG. 4A-4H graphs the results of in vivo survival studies using heterologous transplants from three AML patients generated from FLT-3 + NPM1 + AML samples, which are anti-amanitin-conjugated anti-. A single dose of the CD45 antibody AbB (“anti-CD45-AM”; 1 mg / kg) has been shown to effectively reduce human leukemia cells and prolong survival in a mouse model. FIG. 4A shows the results of a flow cytometry assay evaluating the cell surface expression of CD45 on CD33 + spleen cells from diseased mice. FIG. 4B-4G shows a single intravenous dose of anti-CD45-AM, IgG1 isotype-AM or vehicle (PBS) (these were administered when 2-5% blasts were found in the blood). The survival curves of various PDX AML mice (4-5 mice / group / AML-PDX model) are shown. FIG. 4H shows that the cell surface expression of CD45 on spleen cells derived from diseased mice was evaluated by flow cytometry. FIGS. 5A-5C graphically show the results of the in vivo cell depletion assay, which show that CD45-AM ADC selectively depletes human HSCs and immune cells in humanized NSG mice. FIG. 5A is a schematic diagram of an in vivo humanized NSG model. FIG. 5B shows anti-CD45-amanitin (“CD45-AM”), unconjugated anti-CD45 antibody (“CD45-naked”), or control non-targeting isotype conformance-ADC (“CD45-naked”). Isotype-AM shows the absolute number of human CD45 + cells in peripheral blood 14 days after injection. FIG. 5C shows the absolute number of human CD34 ⁺ cells in the bone marrow of humanized NSG mice 14 days after dosing. Similar results were obtained for CD34 ⁺ CD90 ⁺ (data not shown). * When CD45-AM was compared with any control group, p <0.05. 6A-6C show the design and results of an in vivo pharmacokinetic study showing that the CD45-AM ADC has a fast half-life pharmacokinetic profile in cynomolgus monkeys. FIG. 6A illustrates an overview of the in vivo cynomolgus monkey model and study design (n = 3 / group, except for n = 2 for 1 mg / kg). 6B and 6C graph the pharmacokinetics of anti-CD45-amanitin (“CD45-AM”) in cynomolgus monkeys. These show that CD45-AM exhibits a non-linear PK, a short half-life suitable for transplantation, and a target-mediated drug disposition (TMDD) PK. As used in FIG. 6B, "TAb" refers to all antibodies used to detect anti-hIgG1, while ADC refers to detecting anti-amanitin. 7A, 7B and 7C graph the results of the in vivo reduction assay. These were evaluated 14 days after administration of CD45-AM, and the anti-CD45 amanitin antibody drug conjugate (CD45-Am) reduced peripheral lymphocytes (Fig. 7A) in vivo, and that of cynomolgus monkeys. It is shown that hematopoietic stem cells (HSC), T-, and B-cells can be reduced in the bone marrow. The data in FIG. 7C represent the data from the passage of time shown in FIG. 7A, specifically, the data one day after administration. 8A-8D show the number of red blood cells (FIG. 8A; ¹⁰⁶ / μL units), the number of platelets (platelet) (FIG. 8B; 103 / μL units) ^, plasma compared to the vehicle control (ie, PBS). The results of the assay to detect alanine aminotransaminase levels (Fig. 8C; U / L units) and plasma bilirubin levels (Fig. 8D; U / mL units) show the results of a fast half-life anti-CD45 amanitin antibody drug conju. The number of days after administration of the gate (CD45-Am) at various doses is shown graphically on the horizontal axis. 9A and 9B show the results of a CD45 epitope mapping study using AbA. FIG. 9A is a diagram showing the AbA interaction site on CD45 together with the amino acid sequence of the region containing the AbA epitope (SEQ ID NO: 39). Contact residues were highlighted as 486R, 493Y, 502T (see SEQ ID NO: 37 for numbering). FIG. 9B shows the CD45 PDB structure (5FMV) showing the epitope site (corresponding to amino acid residue 486-502 (RNGPHERYHLEVEAGNT; SEQ ID NO: 38) of CD45) in black. FIG. 9B is a ribbon / surface representation of the front view (A), the rear view (B), the side view 1 (C), the side view 2 (D), and the top view (E). FIG. 10 shows the sequence alignment of the fibronectin D1 to D4 regions of human CD45R0 (SEQ ID NO: 40) and the corresponding regions of cynomolgus monkey (cyno) CD45 (SEQ ID NO: 41). show. The epitope region of human CD45 to which AbA binds and the corresponding region in cynomolgus monkey CD45 are shown in bold.

[Detailed explanation]
For example, an anti-CD45 antibody useful as a therapeutic agent in treating leukemia or acting as a conditioning agent for transplantation, and a conjugate thereof (antibody drug conjugate; ADC) are described in the present application. Disclose. Therefore, an anti-hematopoietic cell antibody (anti-CD45 antibody) useful for hematopoietic stem cell therapy is included in this application. For example, the antibodies or ADCs of this application are useful in conditioning procedures that prepare a patient for receiving a graft containing hematopoietic stem cells. Such a procedure promotes engraftment of hematopoietic stem cell transplantation. According to the method described in this application, an anti-CD45 ADC, an antibody capable of binding to CD45 (for example, CD45 expressed by hematopoietic cells (eg, hematopoietic stem cells or mature immune cells (eg, T cells))). Alternatively, the patient may be conditioned for hematopoietic stem cell transplantation therapy by administering the antigen-binding fragment thereof to the patient. As described in this application, the anti-CD45 antibody may be covalently conjugated to a cytotoxin to form an antibody drug conjugate (ADC). Administration of an ADC capable of binding to CD45 to patients in need of hematopoietic stem cell transplantation therapy, for example, selectively reduces endogenous hematopoietic stem cells, thereby filling them with exogenous hematopoietic stem cell transplants. The availability of space may facilitate engraftment of hematopoietic stem cell transplants.

The following parts may be administered to patients with cancer or autoimmune disorders, or patients in need of hematopoietic stem cell transplantation therapy to promote engraftment of hematopoietic stem cell transplants. A description of an anti-CD45 antibody or conjugate thereof, as well as a method of administering such a therapeutic agent to a patient (eg, prior to hematopoietic stem cell transplantation) are provided.

Definitions As used in this application, the term "about" means a value that is 5% higher or lower than the value stated.

As used in this application, the term "allogeneic", when used in the context of transplantation, is a cell (or tissue) to be transplanted from a genetically dissimilar donor to a recipient of the same species. Or used to define an organ).

As used in this application, the term "self" refers to a cell or graft of which the donor and recipient are the same subject.

As used in this application, the term "heterologous" refers to cells of different species of donor and recipient.

As used in this application, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin and playing a role in the immune response. Immune cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In certain embodiments, natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. Immune cells may be allogeneic or self.

As used in this application, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), genetically engineered antibodies, and other modified forms of antibodies (eg, limited). Although not, de-immunized antibodies, chimeric antibodies, humanized antibodies, heteroconjugated antibodies [eg, bi-, tri- and quad-specific antibodies, diabodies, triabodies, and tetras. Body], as well as antibody fragments [ie, antibody antigen-binding fragments] [eg, Fab', F (ab') ₂ , Fab, Fv, rlgG, and scFv fragments, etc.], as long as they exhibit the desired antigen-binding activity. Included).

Generally, the antibody comprises a heavy chain and a light chain containing an antigen-binding region. Each heavy chain consists of a heavy chain variable region (abbreviated as HCVR or VH in this application) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (abbreviated as LCVR or VL in this application) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)), interspersed with more conserved regions (called skeletal regions (FRs)). .. Each VH and VL is composed of 3 CDRs and 4 FRs and is arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody is that the immunoglobulin binds to a host tissue or factor (eg, various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q), etc.). , May be mediated.

As used herein, the term "antigen binding fragment" refers to one or more portions of an antibody that retains the ability to specifically bind the target antigen. The antigen-binding function of an antibody can be fulfilled by a fragment of a full-length antibody. The antibody fragment may be, for example, Fab, F (ab') ₂ , scFv, Diabody, Triabody, Affibody, Nanobody, aptamer, or domain antibody. Examples of binding fragments included in the term "antigen binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, one consisting of VL, VH, CL and CH1 domains. Valuable Fragments; (ii) F (ab') ₂ Fragments, Divalent Fragments Containing Two Fab Fragments Linked by Disulfide Crosslinks at Hinge Regions; (iii) Fd Fragments Consisting of VH and CH1 Domains; (iv) Antibodies Fv fragments consisting of single arm VL and VH domains; (v) dAb consisting of VH and VL domains; (vi) dAb fragments consisting of VH domains (eg Ward et al., Nature 341: 544-546, 1989). See); (vii) dAb consisting of VH or VL domain; (viii) isolated complementarity determining regions (CDR); and (ix) two or more that may be optionally linked by a synthetic linker (ix) For example, a combination of 2, 3, 4, 5, or 6) isolated CDRs. Furthermore, the two domains (VL and VH) of the Fv fragment are encoded by separate genes, but they are linked by a linker by using a recombinant method, resulting in a single protein chain. (The VL and VH regions pair to form a monovalent molecule), known as a single chain Fv (scFv); for example, Bird et al., Science 242: 423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and said fragments can be screened for usefulness in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, chemical peptide synthesis procedures known in the art. ..

As used in this application, an "intact" or "full length" antibody is an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Point to.

The term "specifically binding" as used in this application refers to the ability of an antibody (or ADC) to recognize and bind to a specific protein structure (epitope) rather than a protein in general. When an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, non-labeled A) in a reaction comprising labeled "A" and said antibody binds to said antibody. The amount of labeled A is reduced. For example, if an antibody (if labeled) can compete with and leave its target by the corresponding non-labeled antibody, the antibody "specifically binds" to the target. In certain embodiments, the antibody specifically binds to a target (eg, an antigen expressed by hematopoietic stem cells [eg, CD45]); the antibody is at least about 10 ^-4 M, about 10 ^-5 M, about. 10 ^-6 M, about 10 ^-7 M, about 10 ^-8 M, about 10 ^-9 M, about 10 ^-10 M, about 10 ^-11 M, about 10 ^-12 M, or less (less than is about) If you have a K _D for the target, for example 10 ^-13 ), which means a number less than 10 ^-12 , it binds specifically. In one embodiment, K _D is measured according to standard Bio-Layer Interferometry (BLI). However, it should be understood that the antibody can specifically bind to more than one antigen with which the sequence is associated. For example, in certain embodiments, the antibody may specifically bind to both human and non-human (eg, mouse or non-human primate) orthologs of the antigen (eg, CD45). Thus, as used in this application, an antibody that "specifically binds to human CD45" binds to human CD45 (and perhaps CD45 from one or more non-human species), but non-CD45. A protein is intended to refer to an antibody that does not substantially bind. Preferably, the antibody is 1x10 ^-7 M or less KD, 5x10 ^-8 _M or less KD, 3x10 ^-8 _M or less KD, 1x10 ^-8 _M or less KD, or 5x10 ^-9 _M or less. At KD, it binds to human _CD45 .

As used in this application, the term "anti-CD45 antibody" or "antibody that binds to CD45" is sufficient affinity for the antibody to be useful as a diagnostic and / or therapeutic agent targeting CD45. Refers to an antibody that can bind to CD45 by sex. In certain embodiments, the anti-CD45 antibody (or fragment thereof) is an anti-human CD45 (hCD45) antibody (or fragment thereof).

The antibodies of the invention are generally isolated or recombinant. As used in this application, "isolated" refers to a polypeptide (eg, antibody) identified and isolated and / or recovered from a cell or cell culture expressing it. Usually, isolated antibodies are prepared by at least one purification step. Thus, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to CD45 is substantially free of antibodies that specifically bind to antigens other than CD45.

As used in this application, the term "monoclonal antibody" is used from a single clone, including any eukaryote, prokaryote, or phage clone, by any means available or known in the art. Refers to and is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombination, and phage display techniques, or combinations thereof. ..

As used in this application, the term "diabody" refers to a divalent antibody comprising two polypeptide chains, where each polypeptide chain is intramolecular in the VH and VL domains on the same peptide chain. It contains VH and VL domains linked by a linker that is too short to associate (eg, a linker composed of 5 amino acids). With this configuration, each domain is paired with a complementary domain on another polypeptide chain to form a homodimer structure. Thus, the term "triabodies" refers to trivalent antibodies containing three peptide chains, each of which is too short to allow intramolecular association of VH and VL domains within the same peptide chain. It contains one VH domain and one VL domain linked by (eg, a linker composed of 1-2 amino acids). Peptides thus constructed are usually trimericated so that the VH and VL domains of adjacent peptide chains are spatially close to each other in order to fold into their natural structure (eg,). , Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-48, 1993).

As used in this application, the term "bispecific antibody" is an antibody that can bind to two different epitopes that may be on the same or different antigens (eg, monoclonal, eg,). , Deimmunized antibody or humanized antibody). For example, one of the binding specificities can be directed to a hematopoietic stem cell surface antigen, eg, CD45, and the other, such as a receptor or receptor subunit specifically involved in signaling pathways that enhance cell proliferation. , Can specifically bind to various hematopoietic stem cell surface antigens or other cell surface proteins. In some embodiments, its binding specificity may be directed to a unique, non-overlapping epitope on the same target antigen (ie, biparatopic antibody).

As used in this application, the term "complementarity determining regions" (CDRs) refers to hypervariable regions found in both the light and heavy chain variable domains of an antibody. The more highly conserved portion of the variable domain is called the skeletal region (FR). The position of amino acids representing the hypervariable region of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, and these positions may be considered within the hypervariable region under one set of criteria, while others. Under the criteria of the set, it may be considered to be outside the hypervariable region. One or more of these positions may also be found in the extended hypervariable region. The antibodies described in this application may contain modifications in these hybrid hypervariable positions. The variable domains of the natural heavy and light chains each have a predominantly β-sheet structure and are linked by three CDRs (CDRs link the β-sheet structures and in some cases form part of them). , Forming a loop), including four skeletal regions. The CDRs in each strand are grouped together closer by the skeletal region in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with the CDRs from other antibody strands, the target binding site of the antibody. (See Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD., 1987). In certain embodiments, immunoglobulin amino acid residue numbering is according to Kabat et al.'S immunoglobulin amino acid residue numbering system (but without limitation, IMGT) unless otherwise noted. And any antibody numbering scheme, including Chothia et al.).

Also, a "conservative sequence modification" of the sequence set forth in the SEQ ID NO: described in this application, i.e., a modification of a nucleotide sequence and an amino acid sequence, encoded by or said above. Also provided is that the binding of the antibody containing the amino acid sequence to the antigen is not lost. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. Modifications may be introduced into the SEQ ID NOs of this application by conventional techniques known in the art, such as site-specific mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues with similar side chains is defined in the art. These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, etc.). Tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, eg) Amino acids having tyrosine, phenylalanine, tryptophan, histidine) can be mentioned. Therefore, preferably, the predicted non-essential amino acid residue in the anti-CD45 antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not disrupt antigen binding are well known in the art (eg, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

The terms "Fc", "Fc region", "Fc domain", and "IgG Fc domain", as used in this application, are immunoglobulins (eg, eg,) relating to a crystallizable fragment obtained by papain digestion of an IgG molecule. It refers to a part of IgG molecule). The Fc region contains the C-terminal half of the two heavy chains of an IgG molecule linked by a disulfide bond. It has no antigen-binding activity, but contains a sugar moiety as well as a binding site for the Fc receptor, including complement and FcRn receptors (see below). For example, the Fc region contains a second constant domain CH2 (eg, residues in EU positions 231 to 340 of human IgG1) and a third constant domain CH3 (eg, residues in EU positions 341 to 447 of human IgG1). include. As used in this application, the Fc region comprises a "lower hinge region" (eg, residues of EU positions 233-239 of human IgG1).

Fc may refer to this isolated region, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at many locations within the Fc domain, including, but not limited to, EU positions 270, 272, 312, 315, 356, and 358, and are therefore presented in this application. There may be slight differences between the sequences to be made and the sequences known in the art. Thus, "wild-type IgG Fc domain" or "WT IgG Fc domain" refers to any naturally occurring IgG Fc region (ie, any allele). Heavy chain sequences of human IgG1, IgG2, IgG3 and IgG4 can be found in many sequence databases, for example, in the Uniprot database (www.uniprot.org) access numbers P01857 (IGHG1_human), P01859, respectively. (IGHG2_human), P01860 (IGHG3_human) and P01861 (IGHG1_human).

As used in this application, the term "modified Fc region" or "variant Fc region" includes one or more amino acid substitutions, deletions, insertions or modifications introduced at any position within the Fc region. Refers to the IgG Fc domain. In certain embodiments, the variant IgG Fc domain results in reduced or no binding affinity for Fc gamma R and / or C1q compared to wild-type Fc domains that do not contain one or more amino acid substitutions. , Contains one or more amino acid substitutions. In addition, Fc binding interactions are a variety of effector functions and downstream signaling events (antibody dependent cell-mediated cytotoxicity (ADCC)) and complement dependent cytotoxicity (complement dependent cytotoxicity (ADCC)). It is essential for CDC)), etc., but not limited to these). Thus, in certain embodiments, an antibody comprising a variant Fc domain (eg, an antibody, fusion protein, or conjugate) does not contain one or more amino acid substitutions, deletions, insertions, or modifications (eg, Fc regions). At least one or more Fc ligands (unmodified Fc regions) containing naturally occurring amino acid residues at the corresponding positions in which have the same amino acid sequence otherwise, compared to the corresponding antibody. For example, the binding affinity for Fc gamma R) may change.

The variant Fc domains described in this application are defined according to the amino acid modifications that make them up. For all amino acid substitutions discussed in this application with respect to the Fc region, the numbering always follows the EU index as in Kabat. Thus, for example, D265C is an Fc variant of the parent Fc domain in which aspartic acid (D) at EU position 265 is replaced with cysteine (C). Note that the order in which the substitutions are indicated is arbitrary. Similarly, for example, D265C / L234A / L235A has substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) for the parent Fc domain. Define a variant. Variants may also be specified according to their final amino acid composition at the mutant EU amino acid position. For example, the L234A / L235A mutation may be referred to as "LALA". As a further example, the E233P.L234V.L235A.delG236 (236 deletion) mutation may be referred to as "EPLVLA del G". As yet another example, the I253A.H310A.H435A mutation may be referred to as "IHH". Note that the order in which the substitutions are indicated is arbitrary.

The term "Fc gamma receptor" or "Fc gamma R" as used in this application refers to any member of the family of proteins that binds to the IgG antibody Fc region and is encoded by the Fc gamma R gene. In humans, this family includes, but is not limited to, Fc gamma RI (CD64) (eg, isoforms Fc gamma RIa, Fc gamma RIb, and Fc gamma RIc, etc.); Fc gamma RII (CD32) (eg, eg). , Isoform Fc gamma RIIa [eg, allotypes H131 and R131], Fc gamma RIIb [eg, Fc gamma RIIb-1 and Fc gamma RIIb-2], and Fc gamma RIIc; and Fc gamma RIII (CD16) (eg, Fc gamma RIIc). , Isoform Fc gamma RIIIa [eg, allotypes V158 and F158] and Fc gamma RIIIb [eg, allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2], and any undiscovered human Fc gamma R or Fc gamma R. Isoforms or allotypes, including Fc gamma R can be derived from any organism, such as, but not limited to, humans, mice, rats, rabbits and monkeys. Mouse Fc gamma R. Fc Gamma RI (CD64), Fc Gamma RII (CD32), Fc Gamma RIII (CD16), and Fc Gamma RIII-2 (CD16-2), as well as any undiscovered mice. Includes Fc gamma R or Fc gamma R isoforms or allotypes.

As used in this application, the term "effector function" refers to a biochemical event resulting from the interaction of an Fc domain with an Fc receptor. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. As used herein, "effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granule lymphocytes, Langerhans cells, natural. Killer (NK) cells, and gamma-delta T cells, and are derived from any organism (eg, but not limited to, humans, mice, rats, rabbits and monkeys).

As used in this application, the terms "silent", "silenced" or "silencing" are compared to the binding of the same antibody containing the unmodified Fc region to FcγR. Thus, it refers to an antibody having a modified Fc region described in this application with reduced binding to the Fc gamma receptor (FcγR) (eg, including the unmodified Fc region when measured by BLI). The reduction in FcγR binding compared to the same antibody binding to FcγR is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. ). In some embodiments, the Fc silenced antibody does not show detectable binding to FcγR. Binding of antibodies with modified Fc regions to FcγR can be performed by various techniques known in the art (eg, but not limited to, equilibrium methods [eg, enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373: 52-60, 2008; Radioimnoassay (RIA)], or surface plasmon resonance assay or kinetic-based assay of other mechanisms [eg, BIACORE ^TM analysis or Octet ^TM analysis (forte BIO) )], And other methods [eg, indirect binding assay, competitive binding assay, fluorescent resonance energy transfer (FRET), gel electrophoresis and chromatography (eg, gel filtration)]). be able to. These and other methods may utilize labels on one or more assessment components and / or various detection methods (eg, but not limited to, color-developing labels, fluorescence). Sex labels, luminescent labels, or isotope labels, etc.) may be used. For binding affinity and kinetics, see Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. It is described in. An example of a competitive binding assay is to incubate a labeled antigen and an antibody of interest in the presence of an increased amount of non-labeled antigen, and to detect an antibody bound to the labeled antigen. Is a radioimmunoassay comprising. From that data, Scatchard plot analysis may determine the affinity and off-rate of the antibody of interest for a particular antigen. Competition with the second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated in the presence of the antibody of interest conjugated to the labeled compound while increasing the amount of the non-labeled second antibody.

As used in this application, the term "same antibody containing unmodified Fc region" lacks the listed amino acid substitutions (eg, D265C, H435A, L234A, and / or L235A), but otherwise. Refers to an antibody having the same amino acid sequence as the Fc-modified antibody to be compared.

The term "antibody dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity, in which a polypeptide containing the Fc domain (eg, an antibody) is referred to. It binds to Fc receptors (FcR) present on certain cellular cytotoxic cells (eg, predominantly NK cells, neutrophils, and macrophages), and these cellular cytotoxic effector cells are "targets" carrying the antigen. It becomes specifically bound to "cells" and subsequently damages the target cells with its cellular toxins (Hogarth et al., Nature review Drug Discovery 2012, 11: 313). In addition to antibodies and fragments thereof, other polypeptides containing the Fc domain that have the ability to specifically bind to antigen-carrying target cells (eg, Fc fusion proteins and Fc-conjugated proteins) are cell-mediated cell injury. Is intended to bring about.

For simplicity, cell-mediated cellular cytotoxicity resulting from the activity of a polypeptide containing the Fc domain is also referred to herein as ADCC activity. The ability of any particular polypeptide of the disclosure to mediate the lysis of target cells by ADCC can be assayed. To assess ADCC activity, a polypeptide of interest (eg, an antibody) is added to the target cells together with the immune effector cells, resulting in cytolysis of the target cells. Cell lysis is generally detected by the release of a label (eg, a radioactive substrate, a fluorescent dye or a natural intracellular protein) from the lysed cell. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Specific examples of the in vitro ADCC assay include Bruggemann et al., J. Exp. Med. 166: 1351 (1987); Wilkinson et al., J. Immunol. Methods 258: 183 (2001); Patel et al., J. . Immunol. Methods 184: 29 (1995). Alternatively, or further, ADCC activity of the antibody of interest in vivo (eg, in an animal model as disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95: 652 (1998)). May be evaluated.

As used in this application, the terms "conditioning" and "conditioning" refer to the process of preparing a patient for receiving a graft (eg, a graft containing hematopoietic stem cells). Point to. Such procedures promote engraftment of hematopoietic stem cell transplants (eg, the amount of viable hematopoietic stem cells in a blood sample isolated from a patient after a conditioning procedure and subsequent hematopoietic stem cell transplantation). Is inferred from the continuous increase). The patient may be conditioned in preparation for hematopoietic stem cell transplantation therapy by administering to the patient an ADC, antibody, or antigen-binding fragment thereof capable of binding to CD45 expressed by hematopoietic stem cells according to the method described in this application. be. As described in this application, the antibody is covalently conjugated to a cytotoxin to form a drug-antibody conjugate (also interchangeably referred to herein as an antibody drug conjugate (ADC)). May be gated. Administering an ADC, antibody, or antigen-binding flagmen thereof capable of binding to one or more of the above antigens to patients in need of hematopoietic stem cell transplantation therapy, for example, selectively reduces endogenous hematopoietic stem cells. This may facilitate engraftment of the hematopoietic stem cell implant by creating a space to be filled by the exogenous hematopoietic stem cell implant.

As used in this application, the term "effective amount" or "therapeutically effective amount" is sufficient to achieve the desired result or to have an effect on an autoimmune disease or cancer in a human patient. Refers to the amount of therapeutic agent, eg, anti-CD45 ADC.

As used in this application, the term "half-life" refers to the time it takes for a plasma concentration of an antibody drug in the body to decrease by half or 50% in a subject, eg, a human subject. This 50% reduction in serum concentration reflects the amount of circulating drug.

As used in this application, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are amino acid residues not encoded by the human germline immunoglobulin sequence (eg, mutations introduced by random or site-specific mutagenesis in vitro, or gene rearrangements or bodies in vivo. May include mutations introduced by cell mutations). However, as used in this application, the term "human antibody" is intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, is transplanted onto a human skeletal sequence. Not done. Non-human animals or prokaryotes or eukaryotic cells capable of expressing human immunoglobulin (eg, heavy and / or light chain) genes in which human antibodies have been reconstituted in human cells (eg, by recombinant expression) or functionally. May be produced by nuclear cells. When a human antibody is a single chain antibody, it may contain linker peptides not found in native human antibodies. For example, Fv may contain a linker peptide such as 2 to about 8 glycine or other amino acid residues, which link the variable regions of the heavy chain and the variable regions of the light chain. do. Such linker peptides are considered to be of human origin. Human antibodies may be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies are also unable to express functional endogenous immunoglobulins, but may also be produced using transgenic mice capable of expressing human immunoglobulin genes (eg, PCT Publication Number). WO 1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; US Pat. No. 5,413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016; No. 5,545,806; No. 5,814,318; See Nos. 5,885,793; Nos. 5,916,771; and Nos. 5,939,598).

The term "chimeric antibody" is an antibody (eg, said variable region sequence derived from a rat antibody, said said constant region), wherein the variable region sequence is derived from one species and its constant region sequence is derived from another species. The sequence is intended to refer to an antibody derived from an antibody derived from a human antibody, etc.). Methods for producing chimeric antibodies are known in the art. For example, Morrison, 1985, Science 229 (4719): 1202-7; Oi et al., 1986, BioTechniques 4: 214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S. Pat Nos. 5,807,715; 4,816,567; and 4,816,397.

The term "de-immunized" or "de-immunization" as used in this application refers to wild-type constructs as non-immunogenic in humans. With respect to modifying the original wild-type construct (or parent antibody) by or by making it more attenuated immunogenic. The deimmunized antibody comprises a portion of non-human origin (eg, framework region and / or CDR). As used in this application, the term "deimmunized antibody" refers to an antibody deimmunized by mutation so as not to activate the subject's immune system (eg, Nanus et al., J. Urology). 170: S84-S89, 2003; WO98 / 52976; WO00 / 34317).

The "humanized" form of a non-human (eg, mouse or rat) antibody is an immunoglobulin containing a minimum of sequences derived from a non-human immunoglobulin. In general, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR regions are those of non-human immunoglobulins. Correspondingly, all or substantially all of the FR regions are of human immunoglobulin sequences. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin consensus sequence. Methods of humanizing an antibody are known in the art. For example, Riechmann et al., 1988, Nature 332: 323-7; US Patent 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al .; EP239400; PCT Published WO 91/09967; US Patent 5,225,539; EP592106; EP519596 Padlan, 1991, Mol. Immunol., 28: 489-498; Studnicka et al., 1994, Prot. Eng. 7: 805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91: See 969-973; and US Pat. No. 5,565,332.

As used in this application, the term "engraftment potential" is used to refer to the ability of hematopoietic stem cells and hematopoietic progenitor cells to regenerate into tissue, such cells circulating naturally. It does not matter whether it is provided by transplantation or by transplantation. The term includes all events surrounding or leading to engraftment, such as tissue homing of cells and colonization of cells, within the tissue of interest. Engraftment efficiency or engraftment rate can be assessed or quantified using any clinically acceptable parameters known to those of skill in the art, eg, competitive repopulating unit (CRU). ); Marker uptake or expression in tissue that has been homing, colonized, or engrafted by stem cells; or disease progression, survival of hematopoietic stem cells and hematopoietic progenitor cells, or recipes. Evaluating the progress of a subject by prolonging the life of the ent can be mentioned. Engraftment can also be determined by measuring the number of leukocyte cells in the peripheral blood after transplantation. Engraftment can also be assessed by measuring the recovery rate of bone marrow cells by donor cells in the bone marrow aspiration sample.

As used in this application, the term "hematopoietic stem cell"("HSC") refers to the ability to self-replicate and granulocytes (eg, promyelinocytes, neutrophils, eosinophils, eosinophils), erythrocytes. (Eg, reticulated erythrocytes, erythrocytes), platelets (eg, megakaryocytes, platelet-producing megakaryocytes, platelets), monospheres (eg, monospheres, macrophages), dendritic cells, microglia , Bone cells, and immature blood cells having the ability to differentiate into mature blood cells, including but not limited to various lineages including but not limited to NK cells, B cells and T cells. Such cells may contain CD34 ⁺ cells. CD34 ⁺ cells are immature cells that express CD34 cell surface markers. In humans, CD34 + cells are thought to contain a subpopulation of cells with the stem cell characteristics defined above, whereas in mice, the HSC is CD34-. Furthermore, HSC also refers to long-term regenerated HSC (LT-HSC) and short-term regenerated HSC (ST-HSC). LT-HSC and ST-HSC are distinguished based on their functional potential and expression of cell surface markers. For example, human HSC is for mature series markers including CD34 +, CD38-, CD45RA-, CD90 +, CD49F +, and lin- (CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A, etc. Negative). In mice, bone marrow LT-HSC is CD34-, SCA-1 +, C-kit +, CD135-, Slamfl / CD150 +, CD48-, and lin- (Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra). On the other hand, ST-HSC is CD34 +, SCA-1 +, C-kit +, CD135-, Slamfl / CD150 +, and lin- (Ter119, CD11b, Gr1, CD3, etc.) Negative for mature sequence markers including CD4, CD8, B220, IL7ra, etc.). In addition, ST-HSC is less homeostatic and more proliferative than LT-HSC under homeostasis conditions. However, LT-HSCs are more self-replicating (ie, LT-HSCs survive throughout adulthood and can be continuously transplanted through recipients), whereas ST-HSCs. , Self-renewal ability is limited (ie, ST-HSC survives for a limited period of time and cannot be continuously transplanted). Any of these HSCs can be used in the methods described in this application. ST-HSCs are particularly useful because they are highly proliferative and therefore can produce differentiated progeny more rapidly.

As used in this application, the term "functional potential of hematopoietic stem cells" is 1) multi-potency (which, but is not limited to, granulocytes [eg, anterior bone marrow]. Spheres, neutrophils, eosinophils, basal spheres], erythrocytes [eg, reticulated erythrocytes, erythrocytes], thrombocytes [eg, meganuclear blasts, platelet-producing macronuclear cells, platelets], Ability to differentiate into multiple different blood lines, including monospheres [eg, monospheres, macrophages], dendritic cells, microglia, osteopathic cells, and lymphocytes [eg, NK cells, T cells and B cells]. ), 2) Self-renewal (which refers to the ability of hematopoietic stem cells to give rise to daughter cells with the same potential as the mother cell, and this ability can occur repeatedly throughout individual survival without exhaustion. ), And 3) the function of hematopoietic stem cells, including the ability to home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis when hematopoietic stem cells or their progeny are reintroduced into the transplant recipient. Refers to typical characteristics.

As used in this application, the terms "subject" and "patient" refer to a human-like organism that is treated for a particular disease or condition as described in this application. For example, patients such as human patients may be treated prior to hematopoietic stem cell transplantation therapy to promote extrinsic hematopoietic stem cell engraftment.

As used in this application, the term "donor" refers to a human or animal in which one or more cells have been isolated prior to administration of the cells or progeny to the recipient. The one or more cells may be, for example, a population of hematopoietic stem cells.

As used in this application, the term "recipient" refers to a patient receiving a graft, such as a graft containing a population of hematopoietic stem cells. The transplanted cells administered to the recipient may be, for example, autologous, allogeneic, or allogeneic cells.

As used in this application, the term "intrinsic" refers to molecules, cells, tissues, or organs (eg, hematopoietic stem cells, or macronuclear cells, platelets) that are naturally found in certain organisms such as human patients. ), Platelets, erythrocytes, mast cells, myeoblasts, basal spheres, neutrophils, eosinophils, microglial cells, granulocytes, monospheres, osteoclasts, antigen-presenting cells, macrophages, Describes substances such as dendritic cells, natural killer cells, T-lymphocytes, or cells of the hematopoietic lineage such as B-lymphocytes).

As used in this application, the term "sample" refers to a sample taken from a subject (eg, blood, blood components (eg, serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (eg, placenta). Or dermal), pancreatic fluid, chorionic villi sample, and cells).

As used in this application, the phrase "substantially cleared from blood" is the time after administration of a therapeutic agent (eg, an anti-CD45 antibody, or an antigen-binding fragment thereof) to a patient, said. When the concentration of the therapeutic agent in a blood sample isolated from a patient is such that the therapeutic agent cannot be detected by conventional methods (eg, the therapeutic agent is used to detect the therapeutic agent). (When the noise threshold of the device or assay is not detected). Antibodies, antibody fragments, and proteins using various techniques known in the art, such as ELISA-based detection assays known in the art or described in this application. May detect ligand. Further assays that can be used to detect antibodies, or antibody fragments, include, among other things, immunoprecipitation and immunoblot assays known in the art.

As used in this application, the terms "to treat" or "treatment" refer to reducing the severity and / or frequency of disease symptoms, disease symptoms and /. Or to eliminate the root cause of the symptoms, reduce the frequency or possibility of the disease symptoms and / or reduce the root cause thereof, and improve or repair the damage directly or indirectly caused by the disease. That refers to any improvement in any outcome of the disease (eg, longer survival, more reduced morbidity and / or reduced side effects that are a by-product of alternative therapeutic modalities); Complete eradication of the disease is preferred, as is readily understood in the art, but this is not a requirement for therapeutic action. Beneficial or desired clinical outcomes, but not limited to, the survival of exogenous hematopoietic cells in patients after antibody conditioning therapy as described in this application followed by hematopoietic stem cell transplantation therapy. It includes the promotion of wearing. Further beneficial consequences are an increase in the number of hematopoietic stem cells or relatives in patients in need of hematopoietic stem cell transplantation after conditioning therapy followed by administration of an extrinsic hematopoietic stem cell transplant to the patient. Concentration increases. The beneficial results of the therapies described in this application also include conditioning therapy followed by hematopoietic stem cell transplantation therapy followed by one or more cells of the hematopoietic lineage (macrocytosis, thrombocytes, platelets). Red blood cells, mast cells, myeloid blasts, basal spheres, neutrophils, eosinophils, microglial cells, granulocytes, monospheres, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T It may also include an increase in the number of cells (such as lymphocytes or B lymphocytes) or an increase in relative concentration. Further beneficial results include cancer cells (eg, CD45 + leukemia cells) or autoimmune cells (eg, CD45 + autoimmune lymphocytes such as CD45 + T-cells that express T-cell receptors that cross-react with self-antigens). It may include a decrease in the amount of disease-causing cell population, such as a population of. As long as the methods of the present disclosure are intended to prevent disability, it is understood that the term "prevent" does not require complete arrest of the disease state. Rather, as used in this application, the term preventive means that one of ordinary skill in the art can identify a population susceptible to a disorder and administer the compounds of the present disclosure prior to the onset of the disease. The term does not mean that the condition of the disease is completely avoided.

As used in this application, patients who "need" for hematopoietic stem cell transplantation are those who show defects or deficiencies in one or more blood cell types, as well as stem cell disorders, autoimmune diseases, cancer, or this application. Includes patients with other pathologies described in. Hematopoietic stem cells are generally 1) multi-potency, and thus, but not limited to, granulocytes (eg, promyelinocytes, neutrophils, eosinophils, basal platelets), erythrocytes. (Eg, reticulated erythrocytes, erythrocytes), platelets (eg, megakaryocytes, platelet-producing megakaryocytes, platelets), monospheres (eg, monospheres, macrophages), dendritic cells, microglia Can differentiate into multiple different blood lines, including, bone-breaking cells, and lymphocytes (eg, NK cells, B-cells and T-cells), 2) self-renewal, and thus equivalent to mother cells. Can give rise to daughter cells with the potential for, and 3) show the ability to home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis when reintroduced into the transplant recipient. .. Therefore, patients who are defective or deficient in one or more cell types of the hematopoietic lineage may be administered hematopoietic stem cells in vivo to reconstitute a population of defective or deficient cells. For example, the patient may have cancer and the deficiency is caused by administration of chemotherapeutic agents or other agents that selectively or non-specifically reduce the cancerous cell population. May occur. Further, or as an alternative, the patient suffers from sickle cell anemia, thalassemia, fanconi anemia, aplastic anemia, and abnormal hemoglobinopathy such as Wiskott-Aldrich syndrome (eg, non-malignant abnormal hemoglobinopathy). There may be. The subject may be a subject suffering from adenosine deaminase severe combined immunodeficiency disease (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, diamond-blackfan anemia, and Schwakmann-diamond syndrome. .. The subject may have or are affected by a hereditary blood disorder (eg, sickle cell anemia) or an autoimmune disease. Further, or as an alternative, the subject may have or be affected by a malignant tumor such as neuroblastoma or hematological malignancies. For example, the subject may have leukemia, lymphoma or myeloma. In some embodiments, the subject is acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelogenous tumors, diffuse large B-cell lymphoma or non-Hodgkin's lymphoma. Has (non-Hodgkin's lymphoma). In some embodiments, the subject has myelodysplastic syndrome. In some embodiments, the subject is an autoimmune disease such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type I diabetes, or another autoimmune condition described in this application. Have. In some embodiments, the subject requires chimeric antigen receptor T-cell (CART) therapy. In some embodiments, the subject has or is affected by a metabolic storage disorder. The subject benefits from metabolic disorders selected from the group consisting of glycogen accumulation, mucopolysaccharidosis, Gauche's disease, Harler's disease, sphingolipidosis, metachromatic leukodystrophy, or the treatments and therapies disclosed in this application. Any other disease or disorder that can be obtained (but not limited to severe complex immunodeficiency, Wiscot-Aldrich syndrome, hyperimmunoglobulin M syndrome, Chediac-East disease, hereditary lymphohistiocytosis) Bone Marrow Transplantation for Non -Malignant disease ", ASH Education Book, 1: 319-338 (2000) (This disclosure relates to pathological conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy, which is in its entirety. Have or are affected by the diseases or disorders described in (incorporated by reference). Further or instead, patients who "need" hematopoietic stem cell transplantation are those who have or do not have one of the aforementioned pathologies (nevertheless, giant nuclei, platelets, Platelets, erythrocytes, mast cells, myeloid blasts, basal cells, neutrophils, eosinocytes, microglia, granulocytes, monospheres, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural cells. Decreased levels of one or more endogenous cell types within the hematopoietic lineage, such as killer cells, T-lymphocytes, and B-lymphocytes [eg, compared to levels of otherwise healthy subjects. Decrease]. Some of those skilled in the art will use, for example, flow cytometry and fluorescence activated cell selection (FACS) methods, among other procedures known in the art, for the above-mentioned cell types or other types of blood cells. It can be easily determined whether the levels of one or more of them are reduced for subjects who are otherwise healthy.

As used in this application, the phrase "stem cell injury" is used by conditioning the target tissue of interest and / or by removing an endogenous stem cell population in the target tissue (eg, from the myeloid tissue of interest). Any disease, disorder, or disease that can be treated or cured by (by removing the endogenous hematopoietic stem cell population or hematopoietic precursor cell population) and / or by engrafting or transplanting stem cells into the target tissue of interest. Broadly refers to symptoms. For example, type I diabetes has been shown to be cured by hematopoietic stem cell transplantation and can benefit from conditioning according to the compositions and methods described in this application. Further disorders that can be treated using the compositions and methods described in this application are, but are not limited to, sickle red blood cell anemia, thalassemia, fanconi anemia, aplastic anemia, Wiscot-Aldrich syndrome. , ADA SCID, HIV / AIDS, allogeneic white dystrophy, diamond-blackfan anemia, and Schwakmann-diamond syndrome. Further disorders that can be treated using the methods of patient conditioning and / or hematopoietic stem cell transplantation described in this application include hereditary blood disorders (eg, sickle erythrocyte anemia) and scleroderma, multiple sclerosis. , Ulcerative colitis, and autoimmune diseases such as Crohn's disease. Further diseases that can be treated using the conditioning and / or transplantation methods described in this application include malignant tumors (eg, neuroblastoma) or hematological cancers (eg, leukemia, lymphoma, and myeloma). Can be mentioned. For example, the cancer may be acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelogenous tumors, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Hodgkin's lymphoma). Further diseases that can be treated using the conditioning and / or transplantation methods described in this application include myelodysplastic syndrome. In some embodiments, the subject has or is affected by a metabolic storage disorder. For example, the subject may be a metabolic disorder selected from the group consisting of glycogen accumulation, mucopolysaccharidosis, Gauche's disease, Harler's disease, sphingolipidosis, metachromatic white dystrophy, or the treatments and therapies disclosed in the present application. Any other disease or disorder that can benefit from (but not limited to severe complex immunodeficiency, Wiscot-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediac-East disease, Hereditary lymphohistiocytosis, marble bone disease, osteodysplasia, storage disease, salacemia major, sickle erythema, systemic sclerosis, systemic lupus erythematosus, polysclerosis, juvenile joints Rheumatoid arthritis), as well as "Bone Marrow Transplantation for Non-Malignant disease", ASH Education Book, 1: 319-338 (2000) (this disclosure relates to pathological conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy. And may be suffering from or affected by any of the diseases or disorders described in (incorporated by reference in this application) in its entirety.

As used in this application, the term "vector" includes nucleic acid vectors such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. Expression vectors described in this application include polynucleotide sequences and, for example, additional sequence elements used to express proteins and / or integrate these polynucleotide sequences into the genome of mammalian cells. May include. Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences such as promoter regions and enhancers that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments include polynucleotide sequences that enhance the translation rate of these genes or improve the stability or transnuclear translocation of mRNA resulting from gene transcription. .. These sequence elements may include, for example, 5'and 3'untranslated regions and polyadenylation signaling sites that allow efficient transcription of genes on expression vectors. Expression vectors described in this application may also include polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

As used in this application, the term "conjugate" or "antibody drug conjugate" or "ADC" refers to an antibody bound to a cytotoxin. ADC is formed by chemical binding of a reactive functional group of one molecule, such as an antibody or antigen-binding fragment thereof, to a suitable reactive functional group of another molecule, such as the cytotoxin described in this application. Will be done. Conjugates may contain a linker between two molecules bound to each other (eg, between an antibody and a cytotoxin). Examples of linkers that can be used to form conjugates include peptide-containing linkers (eg, linkers containing naturally occurring or non-naturally occurring amino acids, such as D-amino acids). Linkers may be prepared using various strategies described in this application and known in the art. Depending on the reactive components in it, the linker can be used, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, etc. Alternatively, it may be cut by organic metal cutting (see, for example, Leriche et al., Bioorg. Med. Chem., 20: 571-582, 2012).

As used in this application, "drug-to-antibody ratio" or "DAR" refers to the number of drugs (eg, amatoxin) bound to the conjugate antibody. Depending on the number of linking sites on the antibody, higher loading numbers are possible, but the ADC DAR may be in the range 1-8. In certain embodiments, the conjugate has a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.

As used in this application, the term "microtubule binder" refers to a compound that acts by disrupting the microtubule network essential for mitosis and interphase cell function in cells. Examples of microtubule binding agents include, but are not limited to, maytasine, maytansinoids, and derivatives thereof (such as those described in this application or known in the art). Vinca alkaloids such as vincristine, vincristine sulfate, vincristine, vincristine sulfate, bindesin, and binorelvin, taxanes such as docetaxel and paclitaxel, macrolides such as discodelmolide, colchicine and epothilone, and derivatives thereof (epotilone B or Its derivatives, etc.).

As used in this application, the term "amatoxin" is a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms, or variants or derivatives thereof (eg, variants or derivatives thereof that may inhibit RNA polymerase II activity). Refers to. Examples of amatoxins useful in combination with the compositions and methods described in this application include, but are not limited to, compounds of formulas (IV), (IVA), (IVB) and (IVC), or. Examples include compounds such as other amatoxins such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanuric acid, or proamanitin. As described in this application, amatoxin may be conjugated to an antibody or antigen-binding fragment thereof, for example, via a linker partial structure (L) (thus forming an ADC). Methods and linkers for conjugating exemplary amatoxins useful for such processes are described below. Exemplary linker-containing amatoxins useful for conjugating to antibodies, or antigen-binding fragments, according to the compositions and methods are also described in this application.

The term "acyl" used in this application refers to -C (= O) R, where, as defined in this application, R is hydrogen ("aldehyde"), C ₁ -C ₁₂ alkyl, C. ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₂₀ aryl, 5-10 member heteroaryl, or 5-10 member heterocyclyl. Non-limited examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.

As used in this application, the term "C ₁ -C ₁₂ alkyl" refers to a linear or branched saturated hydrocarbon having 1-12 carbon atoms. Typical C ₁ -C ₁₂ alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl. On the other hand, the branched C ₁ -C ₁₂ alkyl includes, but is not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. The alkyl groups of C ₁ -C ₁₂ may be unsubstituted or substituted.

The term "alkenyl" as used in this application is a normal, secondary or tertiary carbon atom having at least one site of unsaturatedity, namely a carbon-carbon, sp ² double bond. Refers to C ₂ -C ₁₂ hydrocarbons containing. Examples include, but are not limited to: ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isopentenyl, -1-pentenyl, -2-pentenyl, -3-. Examples thereof include methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl and the like. The alkenyl group may be unsubstituted or substituted.

As used in this application, "alkynyl" is a C ₂ containing at least one site of unsaturatedity, namely a normal, secondary or tertiary carbon atom with a carbon-carbon, sp triple bond. -C ₁₂ Refers to hydrocarbons. Examples include, but are not limited to, acetylene and propargyl. The alkynyl group may be unsubstituted or substituted.

As used in this application, "aryl" refers to a C ₆ -C ₂₀ carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthrasenyl. Aryl groups may be unsubstituted or substituted.

As used in this application, "arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom (typically a terminal or sp ³ carbon atom) is substituted with an aryl radical. .. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethane-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2 -Naphtylethene-1-yl, naphthobenzyl, 2-naphthophenylethane-1-yl and the like. The arylalkyl group contains 6 to 20 carbon atoms, for example, the alkyl moiety of the arylalkyl group (eg, alkenyl, alkenyl, alkynyl group, etc.) is 1 to 6 carbon atoms and the aryl moiety. Is 5 to 14 carbon atoms. Alkaline groups may be unsubstituted or substituted.

As used in this application, "cycloalkyl" refers to saturated carbocyclic radicals, which may be monocyclic or bicyclic. Examples of the cycloalkyl group include a ring having 3 to 7 carbon atoms as a monocycle and a ring having 7 to 12 carbon atoms as a bicycle. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

As used in this application, "cycloalkenyl" refers to unsaturated carbocyclic radicals, which may be monocyclic or bicyclic. Examples of the cycloalkenyl group include a ring having 3 to 6 carbon atoms as a monocycle and a ring having 7 to 12 carbon atoms as a dicyclic ring. Examples of monocyclic cycloalkenyl groups are 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl. , And 1-cyclohexa-3-enyl. The cycloalkenyl group may be unsubstituted or substituted.

As used in this application, "heteroaralkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom (typically a terminal or sp ³ carbon atom) is replaced with a heteroaryl radical. Point. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl and the like. The heteroarylalkyl group contains 6 to 20 carbon atoms, for example, the alkyl moiety of the heteroarylalkyl group (including alkenyl, alkenyl, alkynyl group, etc.) is 1 to 6 carbon atoms, and hetero. The aryl moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety of the heteroarylalkyl group may be a monocycle with 3-7 ring members (2-6 carbon atoms) or 7-10 ring members (4-9). It may be a dicyclic having a carbon atom of (1 to 3 heteroatoms selected from N, O, P, and S) (eg: dicyclic [4,5], [5,5], [5,6], or [6,6] system).

As used herein, "heteroaryl" and "heterocycloalkyl" refer to aromatic or non-aromatic ring systems, respectively, where one or more ring atoms are heteroatoms such as nitrogen, oxygen, and sulfur. Is. Heteroaryl or heterocycloalkyl radicals contain 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. A heteroaryl or heterocycloalkyl is a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S). May be a dicycle with 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) (For example: two rings [4,5], [5,5], [5,6], or [6,6] system). Heteroaryl and heterocycloalkyl may be unsubstituted or substituted.

Heteroaryl and heterocycloalkyl groups are Paquette, Leo A .; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry. of Heterocyclic Compounds, A series of Monographs "(John Wiley & Sons, New York, 1950 to present), especially volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: It is described in 5566.

Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, and the like. Isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridadinyl, indridinyl, isoindrill, 3H-indrill, 1H-indazolyl, prynyl, 4H-quinolinidinyl, phthalazinyl, naphthyldinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridylinyl Examples thereof include le, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazine, frazanyl, phenoxadinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazoridinyl, pyrazolinyl, benzotriazolyl, benzoisoxazolyl, and isatinoyl.

Examples of heterocycloalkyls include, but are not limited to, dihydropyryl, tetrahydropyran (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydropyranyl, tetrahydropyranyl, Included are bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinucridinyl, and morpholinyl.

By way of example, but not limited to, carbon-bonded heteroaryls and heterocycloalkyls are pyridine at the 2, 3, 4, 5, or 6 position, pyridazine at the 3, 4, 5, or 6 position, pyrimidine at the 2, 4, 5th or 6th position, 2nd, 3rd, 5th or 6th position of pyrazine, 2,3,4, or 5th position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, 2,4 of oxazole, imidazole or thiazole , Or 5th place, isoxazole, pyrazole, or isothiazole 3,4, or 5th place, aziridin 2nd or 3rd place, azetidine 2,3 or 4th place, quinoline 2,3,4,5,6 , 7, or 8 or at 1, 3, 4, 5, 6, 7, or 8 of isoquinoline. More typically, carbon-bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, Included are 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example, but not limited to, nitrogen-bound heteroaryls and heterocycloalkyls include aziridine, azetidine, pyrrole, pyrroline, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazolin, 3-imidazolin, pyrazoline, pyrazoline. , 2-Pyrazoline, 3-Pyrazoline, Piperidine, Piperazin, Indole, Indoline, 1H-Indazole 1st, Isoindole or Isoindoline 2nd, Morphorin 4th, and Carbazole or Beta Carbolin 9th. Join. More typically, nitrogen-bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

As used in this application, as well as applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc., "substituted" is one or more hydrogens. It means that each atom is independently substituted with a substituent. Typical substituents are, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH ₂ , -NHR, -N (R) ₂ , -N ^+. (R) ₃ , -CX ₃ , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO ₂ , -N ₃ , -NC (= O) H, -NC (= O) R , -C (= O) H, -C (= O) R, -C (= O) NH ₂ , -C (= O) N (R) ₂ , -SO _3- , -SO ₃ H, -S (= O) ₂ R, -OS (= O) ₂ OR, -S (= O) ₂ NH _2, -S (= O) ₂ N (R) ₂ , -S (= O) R, -OP ( = O) (OH) _2, -OP (= O) (OR) ₂ , -P (= O) (OR) ₂ , -PO ₃ , -PO ₃ H ₂ , -C (= O) X, -C (= S) R, -CO ₂ H, -CO ₂ R, -CO _2- , -C (= S) OR, -C (= O) SR, -C (= S) SR, -C (= O) ) NH _2, -C (= O) N (R) ₂ , -C (= S) NH _2, -C (= S) N (R) ₂ , -C (= NH) NH _2, and -C ( = NR) N (R) ₂ , where each X is independently selected from F, Cl, Br, and I for each case; and each R is C ₁ -C. ₁₂ Alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₄ heterocycloalkyl or heteroaryl, protective groups and prodrac moieties are independently selected for each case. In any case where a group is described as "optionally substituted", the group may be independently substituted with one or more of the above substituents in each case.

It is important to understand that certain radical naming conventions can include either mono-radicals or di-radicals, depending on the circumstances. For example, if a substituent requires the position of two bonds to the rest of the molecule, the substituent is understood to be a di-radical. For example, di-radicals such as -CH _2- , -CH ₂ CH _2- , -CH ₂ CH (CH ₃ ) CH _2- , etc. are substituents identified as alkyls that require two bond positions. Can be mentioned. Other radical naming conventions clearly indicate that the radical is a di-radical such as "alkylene", "alkenylene", "allylen", "heterocycloalkylene" and the like.

Wherever a substituent is shown as a di-radical (ie, has two bonding positions to the rest of the molecule), the substituent should be attached in any directional arrangement unless otherwise noted. Please understand that you can.

Anti-CD45 antibody can bind to both human and cynomolgus monkey CD45, and can be used as a therapeutic agent alone or as an antibody drug conjugate (ADC), eg, (i) a cancer characterized by CD45 + cells. And anti-CD45 antibodies that can promote the engraftment of transplanted hematopoietic stem cells in patients who treat autoimmune diseases and (ii) in need of transplantation therapy are disclosed in this application. These therapeutic activities are associated, for example, with an isolated anti-CD45 antibody, an antigen-binding fragment thereof, that binds to CD45 expressed on the surface of cells (eg, cancer cells, autoimmune cells, hematopoietic stem cells, etc.). It can be caused by doing so, and then by inducing cell death. Decreasing endogenous hematopoietic stem cells may provide a niche in which transplanted hematopoietic stem cells can be homing, and subsequently, productive hematopoiesis may be established. In this way, the transplanted hematopoietic stem cells can successfully engraft within a patient (eg, a human patient suffering from the stem cell disorder described in this application).

CD45 is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase essential for signal transduction via T and B cell antigen receptors. CD45 has a large extracellular domain and a cytoplasmic domain containing phosphatase. CD45 can act as both positive and negative regulators, depending on the nature of the stimulus and the cell types involved. The CD45 gene has been rearranged in various ways, but so far only six isoforms have been identified in humans. The isoforms are RA (Uniprot accession number: P08575-8; SEQ ID NO: 31), RO (NCBI accession number: NP_563578.2; SEQ ID NO: 32), RB. (NCBI accession number: XP_006711537.1; SEQ ID NO: 33), RAB (NCBI accession number: XP_006711535.1; SEQ ID NO: 34), RBC (NCBI accession number:: XP_006711536.1; SEQ ID NO: 35), and RABC (NCBI accession number NP_002829.3; SEQ ID NO: 36) (Hermiston et al. 2003 “CD45: a critical regulator”. of signaling thresholds in immune cells. ”Annu Rev Immunol. 2: 107-137.). CD45RA is expressed on naive T cells, and CD45RO is expressed on activated and memory T cells, some B cell subsets, activated monocytes / macrophages, and granulocytes. CD45RB is expressed on peripheral B cells, naive T cells, thymocytes, and weakly on macrophages and dendritic cells.

As described below, a novel anti-human CD45 (hCD45) antibody and fragments thereof were identified by immunizing rats with human CD45. The identified antibody has diagnostic and therapeutic properties. Antibody A (AbA), antibody B (AbB), and antibody C (AbC) were identified in this screening. These antibodies cross-react with human CD45 and rhesus monkey CD45. In addition, these antibodies disclosed in this application can bind to the extracellular domain of various isoforms of human CD45. Thus, in certain embodiments, the antibody of the present application is a pan-specific anti-CD45 antibody (ie, an antibody that binds to all six human CD45 isoforms). In addition, AbA, AbB, and AbC (or antibodies with binding regions or specificities for these antibodies) disclosed in this application can also bind to cynomolgus monkey CD45. Although it was difficult to identify both cross-reactive cynomolgus monkey / human anti-CD45 antibodies and pan-specific antibodies to multiple human isoforms of CD45, Example 1 below. Meet by the screening described in.

The amino acid sequences of the various binding regions of the anti-CD45 antibodies AbA, AbB, and AbC are listed in Table 3. Humanized and chimeric anti-CD45 antibodies based on the antibodies AbA, AbB, or AbC (eg, including the CDRs listed in Table 3) are included in the invention.

In certain embodiments, the present invention provides an anti-CD45 antibody, or antigen-binding fragment thereof, comprising a binding region (eg, CDR, variable region) corresponding to the binding region of AbA. The amino acid sequence of the heavy chain variable region (VH) of Ab is shown in SEQ ID NO: 1 (see Table 3). The amino acid sequence of the VH CDR domain of AbA is SEQ ID NO: 2 (VH CDR1); SEQ ID NO: 3 (VH CDR2) and SEQ ID NO: 4 (VH CDR3). Shown in. The amino acid sequence of the light chain variable region (VL) of AbA is set forth in SEQ ID NO: 5 (see Table 3). The amino acid sequence of the VL CDR domain of AbA is SEQ ID NO: 6 (VL CDR1); SEQ ID NO: 7 (VL CDR2) and SEQ ID NO: 8 (VL CDR3). Shown in. Thus, in certain embodiments, the anti-CD45 antibody provided by the present application, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 1. (SEQ ID NO): Contains a light chain variable region comprising the amino acid sequence set forth in 5. In certain embodiments, the anti-CD45 antibody is a heavy chain comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 2, 3, and 4, as well as SEQ ID NO (SEQ ID NO). ): Includes a light chain variable region containing CDR1, CDR2, and CDR3, comprising the amino acid sequences set forth in 6, 7, and 8.

Anti-human CD45 antibodies, or fragments thereof, that bind to an epitope on human CD45 to which any one of the antibodies AbA, AbB, or AbC (or an antibody having a binding region of AbA, AbB, or AbC) binds. , Intended in this application. Anti-human CD45 antibodies, or antigen-binding fragments thereof, that compete with any one of the antibodies AbA, AbB, or AbC (or antibodies having an AbA, AbB, or AbC binding region) are also further contemplated.

を含む領域中のそれに対応する残基で、ヒトCD45に特異的に結合する。いくつかの実施形態では、抗-CD45抗体、又はその抗原結合フラグメントは、ヒトCD45のフィブロネクチン・ドメイン(例えば、フィブロネクチンd4ドメイン)に特異的に結合する。 In some embodiments, the anti-CD45 antibody, or antigen-binding fragment thereof, specifically binds to human CD45 in a region comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38). For example, in certain embodiments, the anti-CD45 antibody, or antigen-binding fragment thereof, is amino acid residue 486R, 493Y of SEQ ID NO: 37 (fragment of the CD45 isoform corresponding to NP_002829.3). , And at 502T, or in other human CD45 isoforms

The corresponding residue in the region containing, specifically binds to human CD45. In some embodiments, the anti-CD45 antibody, or antigen-binding fragment thereof, specifically binds to the fibronectin domain of human CD45 (eg, the fibronectin d4 domain).

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 containing residues 486R, 493Y, and 502T of SEQ ID NO: 37. It also binds to cynomolgus monkeys and / or rhesus monkeys CD45.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38), and cynomolgus monkeys and It also binds to rhesus monkey CD45.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 39), cynomolgus monkeys and It binds to rhesus monkey CD45.

In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 containing residues 486R, 493Y, and 502T of SEQ ID NO: 37. At least one additional amino acid, at least two additional amino acids, at least three additional amino acids, at least four additional amino acids, or at least at least in a peptide containing RNGPHERYHLEVEAGNT (SEQ ID NO: 38). Binds to 5 additional amino acids, where the residue of the additional amino acid is not the residues 486R, 493Y, and 502T of SEQ ID NO: 37; it also binds to cynomolgus monkey and red mullet CD45. ..

In certain embodiments, the present invention provides an anti-CD45 antibody, or antigen-binding fragment thereof, comprising a binding region (eg, CDR, variable region) corresponding to the binding region of AbB. The amino acid sequence of the heavy chain variable region (VH) of AbB is shown in SEQ ID NO: 9 (see Table 3). The amino acid sequence of the VH CDR domain of AbB is SEQ ID NO: 10 (VH CDR1); SEQ ID NO: 11 (VH CDR2) and SEQ ID NO: 12 (VH CDR3). Shown in. The amino acid sequence of the light chain variable region (VL) of AbB is set forth in SEQ ID NO: 13 (see Table 3). The amino acid sequence of the VL CDR domain of AbB is SEQ ID NO: 14 (VL CDR1); SEQ ID NO: 15 (VL CDR2) and SEQ ID NO: 16 (VL CDR3). Shown in. Thus, in certain embodiments, the anti-CD45 antibody provided by the present application, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9, and SEQ ID NO: 9. (SEQ ID NO): Contains a light chain variable region comprising the amino acid sequence set forth in 13. In certain embodiments, the anti-CD45 antibody is a heavy chain comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11 and 12, as well as SEQ ID NO (SEQ ID NO). ): Includes a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in 14, 15, and 16.

In certain embodiments, the present invention provides an anti-CD45 antibody, or antigen-binding fragment thereof, comprising a binding region (eg, CDR, variable region) corresponding to the binding region of AbC. The amino acid sequence of the heavy chain variable region (VH) of AbC is shown in SEQ ID NO: 17 (see Table 3). The amino acid sequence of the VH CDR domain of AbC is SEQ ID NO: 18 (VH CDR1); SEQ ID NO: 19 (VH CDR2) and SEQ ID NO: 20 (VH CDR3). Shown in. The amino acid sequence of the light chain variable region (VL) of AbC is set forth in SEQ ID NO: 21 (see Table 3). The amino acid sequence of the VL CDR domain of AbC is SEQ ID NO: 22 (VL CDR1); SEQ ID NO: 23 (VL CDR2) and SEQ ID NO: 24 (VL CDR3). Shown in. Thus, in certain embodiments, the anti-CD45 antibody provided by the present application, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a SEQ ID NO: (SEQ ID NO): Contains a light chain variable region comprising the amino acid sequence set forth in 21. In certain embodiments, the anti-CD45 antibody is a heavy chain comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, as well as SEQ ID NO (SEQ ID NO). ): Includes a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in 22, 23, and 24.

In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising the HC variable domain described in Table 3, or a variant of the HC variant region of Table 3, which variant is (i) 1. , 2, 3, 4 or 5 amino acid substitutions, additions or deletions differ from the HC variable domains listed in Table 3; (ii) at most 5, 4, 3, 2 or 1 amino acid substitutions, additions or deficiencies. In loss, it differs from the HC variable domains listed in Table 3; (iii) in table 3 for substitutions, additions or deletions of amino acids 1-5, 1-3, 1-2, 2-5 or 3-5. Different from the HC variable domain described and / or at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In any of (i)-(iv), the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution, which comprises an amino acid sequence.

In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising the LC variable domain described in Table 3, or a variant thereof, wherein the variant is (i) 1, 2, 3, 4 Or, in the substitution, addition or deletion of 5 amino acids, it differs from the LC variable domain shown in Table 3; (ii) in the substitution, addition or deletion of at most 5, 4, 3, 2 or 1 amino acid, in Table 3. Different from the LC variable domains described; (iii) with the LC variable domains listed in Table 3 in the substitutions, additions or deletions of amino acids 1-5, 1-3, 1-2, 2-5 or 3-5. Is different and / or (iv) at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the LC variable domains listed in Table 3. In any of (i)-(iv), the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution, which comprises an amino acid sequence.

In certain embodiments, the anti-CD45 antibody comprises the CDRs set forth in Table 3 of the present application, wherein said CDR is the CD45 specificity (ie, AbA, AbB, or AbC-like specificity) of the antibody. Includes conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining sex).

In certain embodiments, the anti-CD45 antibody is an AbA, AbB or AbC antibody, or a de-imunized antibody based on an antigen-binding portion thereof. A deimmunized antibody is one in which its V region has been selected to lack the T-cell epitope or modified to remove the T-cell epitope, whereby the antibody is immunogenic. The likelihood of becoming sex is minimized or eliminated. In certain embodiments, the anti-CD45 antibody is a T-cell epitope (when the T-cell epitope is present in the sequence of said antibody, a human subject undergoes a HAHA / HAMA reaction to the anti-CD45 antibody. By selecting or manipulating the skeletal domain so that it does not include (causing immune-mediated reactions that can cause, resulting in adverse events in human subjects or diminishing therapeutic efficacy). By doing so, it is deimmunized. The antibodies disclosed in this application (ie, the AbA, AbB, and AbC variable sequences and CDR sequences shown in Table 3) may serve as parent sequences from which deimmunized antibodies are derived. Can be made to.

Humanized Anti-CD45 Antibodies The present invention includes humanized anti-CD45 antibodies based on the antibodies AbA, AbB, or AbC described in this application. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from the "imported" variable domain. Humanization basically follows the method of Winter and collaborators (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536), may be performed by substituting the corresponding sequence of human antibody with a hypervariable region sequence. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than the intact human variable domain is replaced by the corresponding sequence from a non-human species (US). Patent No. 4,816,567). In practice, humanized antibodies are typically human antibodies, where some hypervariable region residues and optionally some FR residues are derived from similar sites in rodent antibodies. Substituted by the residue to be.

Choosing the human variable domain (both light and heavy chains) to use in making humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human skeleton for humanized antibodies (Sims et al. (1993) J Immunol. 151: 2296; Chothia et al. (1987) J Mol. Biol. . 196: 901). Another method uses a particular skeleton derived from the consensus sequence of all human antibodies in a particular subgroup of light or heavy chains. The same skeleton may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol. ., 151: 2623).

It is even more important to humanize the antibody while preserving its high affinity for the antigen and other suitable biological properties. To achieve this goal, one method is to obtain humanized antibodies by the process of analyzing parental sequences and various conceptual humanized products while using three-dimensional models for parental and humanized sequences. , Prepare. It is generally possible to obtain a three-dimensional immunoglobulin model and is well known to those of skill in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures for selected candidate immunoglobulin sequences. By examining what they display, it is possible to analyze the role that the residue is likely to play in the functioning of the candidate immunoglobulin sequence (ie, the candidate immunoglobulin binds to its antigen). Analysis of residues that affect the ability to act). In this way, FR residues are selected from the recipient's sequence and the import sequence so as to achieve the desired antibody characteristics (eg, increased affinity for its target antigen). And can be combined. In general, hypervariable region residues are directly and most substantially involved in affecting antigen binding.

Fc-Modified Antibodies The present disclosure comprises antibodies, or antigen-binding fragments thereof, that have Fc modifications that allow Fc silence and are capable of binding to an antigen expressed by hematopoietic cells (eg, CD45, etc.). Transplanted as a single therapeutic agent or as an ADC, (i) for treating cancer and autoimmune diseases characterized by CD45 + hematopoietic stem cells, and (ii) in patients requiring transplantation therapy. It is based in part on the finding that it promotes engraftment of hematopoietic stem cells. These therapeutic activities include, for example, an anti-CD45 antibody, or antigen-binding fragment thereof, which binds to CD45 expressed by blood cells (eg, hematopoietic stem cells, or mature immune cells (eg, T cells)). It may be caused by binding to cells, autoimmune cells, hematopoietic stem cells, etc., and subsequently by inducing cell death. Decreasing endogenous hematopoietic stem cells may provide a niche in which transplanted hematopoietic stem cells can be homing, and subsequently, productive hematopoiesis may be established. In this way, the transplanted hematopoietic stem cells can successfully engraft within a patient (eg, a human patient suffering from the stem cell disorder described in this application). The Fc-modified antibodies and ADCs of this application not only selectively reduce endogenous hematopoietic stem cells, but also reduce the cytotoxic effect on extrinsic hematopoietic stem cell transplants, thereby engrafting hematopoietic stem cell transplants. Is further promoted.

The antibodies or binding fragments described in this application are modifications and / or mutations (eg, increase or decrease in half-life, or increase or decrease ADCC, modifications and / or modifications that alter the properties of the antibody and / or fragment. / Or mutation) may be included.

In certain embodiments, an antibody comprising one or more radiolabeled amino acids is provided. Radiolabeled antibodies can be used for both diagnostic and therapeutic purposes (another feature is the ability to be conjugated to a radiolabeled molecule). Non-limiting examples of polypeptide labeling include, but are not limited to, 3H, 14C, 15N, 35S, 90Y, 99Tc and 125I, 131I and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (eg, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686, 2nd Edition, Chafner and London, ed., Lippincott Raven (1996). ) And US Patent No. 4,681,581, US Patent No. 4,735,210, US Patent No. 5,101,827, US Patent No. 5,102,990 [U.S. RE35,500], US Patent No. 5,648,471, US Patent No. 5,697,902). For example, radioisotopes may be conjugated by the method of chloramine-T.

In certain embodiments, the anti-CD45 antibody, or binding fragment thereof, comprises a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification to the wild Fc region, resulting in. , The molecule changes its affinity or binding to Fc gamma R (FcγR). Certain amino acid positions within the Fc region are known by crystallographic studies in direct contact with FcγR. Specifically, amino acids 234 to 239 (hinge region), amino acids 265 to 269 (B / C loop), amino acids 297 to 299 (C'/ E loop), and amino acids 327 to 332 (F / G) loop. .. (See Sondermann et al, 2000 Nature, 406: 267-273). In some embodiments, the antibodies described in this application have modified variant Fc regions with at least one residue that are in direct contact with FcγR based on structural and crystallographic analysis. May include. In certain embodiments, the Fc region of the anti-CD45 antibody (or fragment thereof) is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991). Includes amino acid substitution at amino acid 265 by EU index as in (incorporated by reference). "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. In certain embodiments, the Fc region comprises a D265A mutation. In certain embodiments, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 by an EU index, eg, as in Kabat.

In certain embodiments, the Fc region comprises mutations at amino acid positions of D265, V205, H435, I253, and / or H310. For example, specific mutations at these positions include D265C, V205C, H435A, I253A, and / or H310A.

In certain embodiments, the Fc region comprises an L234A mutation. In some embodiments, the Fc region of the anti-CD45 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 with an EU index as in Kabat. In certain embodiments, the Fc region comprises an L235A mutation. In yet another embodiment, the Fc region comprises L234A and L235A mutations. In a further embodiment, the Fc region comprises the D265C, L234A, and L235A mutations. In yet further embodiments, the Fc region comprises the D265C, L234A, L235A, and H435A mutations. In a further embodiment, the Fc region comprises the D265C and H435A mutations.

In yet another embodiment, the Fc region comprises L234A and L235A mutations (also referred to herein as "L234A.L235A" or "LALA"). In another embodiment, the Fc region contains the L234A and L235A mutations, where the Fc region does not contain the P329G mutation. In a further embodiment, the Fc region comprises D265C, L234A, and L235A mutations (also referred to herein as "D265C.L234A.L235A"). In another embodiment, the Fc region contains the D265C, L234A, and L235A mutations, where the Fc region does not contain the P329G mutation. In a further embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations (also referred to herein as "D265C.L234A.L235A.H435A"). In another embodiment, the Fc region contains the D265C, L234A, L235A, and H435A mutations, where the Fc region does not contain the P329G mutation. In a further embodiment, the Fc region comprises a D265C and H435A mutation (also referred to herein as "D265C.H435A"). In yet another embodiment, the Fc region comprises D265A, S239C, L234A, and L235A mutations (also referred to herein as "D265A.S239C.L234A.L235A"). In yet another embodiment, the Fc region comprises the D265A, S239C, L234A, and L235A mutations, where the Fc region does not contain the P329G mutation. In another embodiment, the Fc region comprises D265C, N297G, and H435A mutations (also referred to herein as "D265C.N297G.H435A"). In another embodiment, the Fc region comprises the D265C, N297Q, and H435A mutations (also referred to herein as "D265C.N297Q.H435A"). In another embodiment, the Fc region comprises an E233P, L234V, L235A and delG236 (236 deletion) mutation (also referred to herein as "E233P.L234V.L235A.delG236" or "EPLVLA delG"). In another embodiment, the Fc region comprises the E233P, L234V, L235A and delG236 (236 deletion) mutations, wherein the Fc region does not contain the P329G mutation. In another embodiment, the Fc region is also referred to as E233P, L234V, L235A, delG236 (236 deletion) and H435A mutation (in this application, "E233P.L234V.L235A.delG236.H435A" or "EPLVLA del G.H435A". Call) is included. In another embodiment, the Fc region comprises the E233P, L234V, L235A, delG236 (236 deletion) and H435A mutations, wherein the Fc region does not contain the P329G mutation. In another embodiment, the Fc region comprises L234A, L235A, S239C and D265A mutations. In another embodiment, the Fc region contains the L234A, L235A, S239C and D265A mutations, where the Fc region does not contain the P329G mutation. In another embodiment, the Fc region comprises H435A, L234A, L235A, and D265C mutations. In another embodiment, the Fc region comprises the H435A, L234A, L235A, and D265C mutations, where the Fc region does not contain the P329G mutation.

In some embodiments, the antibody has a modified Fc region, so that in the antibody, the binding to the Fc receptor (FcR) in the in vitro effector function assay is an unmodified Fc region. Compared to the binding of the same antibody to said FcR, it is reduced and the effector function is reduced. In some embodiments, the antibody has a modified Fc region, so that the antibody has unmodified Fc binding to the Fc gamma receptor (FcγR) in an in vitro effector function assay. Compared to the binding of the same antibody containing the region to the FcγR, it is reduced and the effector function is reduced. In some embodiments, the FcγR is FcγR1. In some embodiments, the FcγR is FcγR2A. In some embodiments, the FcγR is FcγR2B. In some embodiments, the FcγR is FcγR2C. In some embodiments, the FcγR is FcγR3A. In some embodiments, the FcγR is FcγR3B. In other embodiments, the reduction in binding is at least 70% reduction, at least 80% reduction in antibody binding to FcγR compared to binding of the same antibody containing the unmodified Fc region to said FcγR. , At least 90% reduction, at least 95% reduction, at least 98% reduction, at least 99% reduction, or 100% reduction. In other embodiments, the reduction in binding is at least 70% to 100% reduction in antibody binding to FcγR, at least 80%, compared to binding of the same antibody containing the unmodified Fc region to said FcγR. % To 100% reduction, at least 90% to 100% reduction, at least 95% to 100% reduction, or at least 98% to 100% reduction.

In some embodiments, the antibody has a modified Fc region, so that the antibody is compared to the cytokine release of the same antibody containing the unmodified Fc region in an in vitro cytokine release assay. , Cytokine release is reduced, with at least 50% reduction in cytokine release. In some embodiments, the reduction in cytokine release is at least 70% reduction, at least 80% reduction, at least 90% reduction in cytokine release compared to cytokine release of the same antibody containing the unmodified Fc region. A decrease, a decrease of at least 95%, a decrease of at least 98%, a decrease of at least 99%, or a decrease of 100%. In some embodiments, the reduction in cytokine release is at least 70% -100% reduction, at least 80% -100% reduction in cytokine release compared to cytokine release of the same antibody containing the unmodified Fc region. Decrease, at least 90% -100% decrease, at least 95% -100% decrease. In certain embodiments, cytokine release is by immune cells.

In some embodiments, the antibody has a modified Fc region so that in the in vitro mast cell degranulation assay, the mast cell degranulation of the same antibody comprising the unmodified Fc region. Compared to, at least 50% mast cell degranulation is reduced and mast cell degranulation is reduced. In some embodiments, the reduction in mast cell degranulation is at least 70% reduction, at least 80% reduction in mast cell degranulation compared to mast cell degranulation of the same antibody containing the unmodified Fc region. Decrease, at least 90% decrease, at least 95% decrease, at least 98% decrease, at least 99% decrease, or 100% decrease. In some embodiments, the reduction in mast cell degranulation is at least 70% to 100% reduction in mast cell degranulation, at least compared to mast cell degranulation of the same antibody containing the unmodified Fc region. 80% -100% reduction, at least 90% -100% reduction, at least 95% -100% reduction.

In some embodiments, the antibody has a modified Fc region, so that the antibody is modified in an antibody dependent cell phagocytosis (ADCP) assay. ADCP is reduced or avoided by at least 50% reduction to antibody-dependent cell phagocytosis (ADCP) compared to ADCP of the same antibody containing no Fc region. In some embodiments, the reduction in ADCP is at least 70% reduction, at least 80% reduction, at least 90% reduction in cytokine release compared to cytokine release of the same antibody containing the unmodified Fc region. , At least 95% reduction, at least 98% reduction, at least 99% reduction, or 100% reduction.

In some embodiments, the anti-CD45 antibody described in this application comprises an Fc region comprising one of the following modifications or combinations of modifications: D265A, D265C, D265C / H435A, D265C / LALA. , D265C / LALA / H435A, D265A / S239C / L234A / L235A / H435A, D265A / S239C / L234A / L235A, D265C / N297G, D265C / N297G / H435A, D265C (EPLVLAdelG *), D265C (EPLVLAdelG) / H435A N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, EPLVLAdelG / D265C, EPLVLAdelG / D265A, N297A, N297G, or N297Q.

The binding or affinity between the modified Fc region and the Fc gamma receptor can be determined by various techniques known in the art (eg, but not limited to, equilibration methods [eg, enzyme-linked immunosorbent assay (eg, enzyme-linked immunosorbent assay). ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373: 52-60, 2008; Radioimnoassay (RIA)], or surface plasmon resonance assay or kinematically based assay of other mechanisms [eg, BIACORE®. ) Analysis or Octet® analysis (forteBIO)], as well as other methods [eg, indirect binding assay, competitive binding assay, fluorescent resonance energy transfer (FRET), gel electrophoresis and chromatography (eg, gel filtration). ) Etc.]) may be used for measurement. These and other methods may utilize labels on one or more components to be evaluated, and / or various detection methods (eg, but not limited to, color-developing labels, Fluorescent labels, luminescent labels, or isotope labels, etc.) may be used. For binding affinity and kinetics, see Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. It is described in. An example of a competitive binding assay is to incubate a labeled antigen and an antibody of interest in the presence of an increased amount of non-labeled antigen, and to detect an antibody bound to the labeled antigen. Is a radioimmunoassay comprising. From that data, Scatchard plot analysis may determine the affinity and off-rate of the antibody of interest for a particular antigen. Competition with the second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated in the presence of the antibody of interest conjugated to the labeled compound while increasing the amount of the non-labeled second antibody.

In certain embodiments, an antibody having an Fc modification described in this application (eg, D265C, L234A, L235A, and / or H435A) contains the same Fc region whose binding to the Fc gamma receptor is unmodified. At least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98% reduction, at least 99% reduction compared to the binding of the antibody to the Fc gamma receptor. , Or a 100% reduction (eg, as assessed by biolayer interferometry (BLI)).

Without wishing to be bound by any theory, Fc region binding interactions with Fc gamma receptors have various effector functions and downstream signaling events (eg, but not limited to, antibody-dependent). It is considered essential for antibody-dependent cellular-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, in certain embodiments, antibodies containing modified Fc regions (eg, including L234A, L235A, and / or D265C mutations) have substantially reduced or abolished effector function. Effector function may be assayed using various methods known in the art (eg, by measuring the cellular response to the antibody of interest [eg, mast cell degranulation or cytokine release]). For example, using standard methods in the art, the Fc-modified antibody is released in vitro by its ability to induce mast cell degranulation, or its cytokine release (eg, by human peripheral blood mononuclear cells). It may be assayed for its ability to induce mast cells.

The antibodies of the present disclosure have been further modified to include, for example, (Dall'Acqua et al. (2006) J Biol Chem 281: 23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004) J Biol Chem 279: 6213-6), (Hinton et al. (2006) J Immunol 176: 346-56), (Shields et al. (2001) J Biol Chem 276: 6591- 604), (Petkova et al. (2006) Int Immunol 18: 1759-69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2005) Nat Biotechnol Fc such as the Fc variants described in 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and (Kim et al. (1999) Eur J Immunol 29: 2819-25). Further introduction of the mutation may further regulate the half-life of the antibody and may include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435. .. Exemplary mutations that can be made alone or in combination are the T250Q, M252Y, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations. ..

Thus, in certain embodiments, the Fc region comprises a mutation that results in a reduced half-life (eg, as compared to an antibody having an unmodified Fc region). Antibodies with short half-lives are described in this application in certain cases where the antibody is expected to function as a short-lived therapeutic agent (eg, administer the antibody followed by HSC). In the conditioning step), it can be beneficial. Ideally, unlike endogenous stem cells, the antibody is substantially expressed prior to administration of HSC, which generally expresses the target antigen (eg, CD45) but is not the target of the anti-CD45 antibody. Should be cleared. In one embodiment, the Fc region comprises a mutation at position 435 (EU index by Kabat). In certain embodiments, the mutation is the H435A mutation.

In certain embodiments, the anti-CD45 antibodies described in this application (eg, in humans) are about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less, about 20 hours or less, about 19. It has a half-life of less than an hour, about 18 hours or less, about 17 hours or less, about 16 hours or less, about 15 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less, or about 11 hours or less.

In certain embodiments, the anti-CD45 antibodies described in this application have a half-life of about 1-5 hours, about 5-10 hours, about 10-15 hours, about 15-20 hours, or about 20-25 hours. Have (eg, in humans). In certain embodiments, the half-life of the anti-CD45 antibody is about 5-7 hours; about 5-9 hours; about 5-11 hours; about 5-13 hours; about 5-15 hours; about 5-20 hours. Approximately 5 to 24 hours; Approximately 7 to 24 hours; Approximately 9 to 24 hours; Approximately 11 to 24 hours; Approximately 12 to 22 hours; Approximately 10 to 20 hours; Approximately 8 to 18 hours; be.

In some embodiments, the Fc region comprises two or more mutations that reduce half-life and reduce the effector function of the antibody. In some embodiments, the Fc region is a mutation that results in a reduced half-life, and at least one residue that can be in direct contact with FcγR (eg, based on structural and crystallographic analysis). Includes mutations in the group. In certain embodiments, the Fc region comprises an H435A mutation, an L234A mutation, and an L235A mutation. In certain embodiments, the Fc region comprises an H435A mutation and a D265C mutation. In certain embodiments, the Fc region comprises an H435A mutation, an L234A mutation, an L235A mutation, and a D265C mutation.

In some embodiments, the antibody, or antigen-binding fragment thereof, is conjugated to a cellular toxin (eg, amatoxin) via a cysteine residue in the Fc domain of the antibody, or antigen-binding fragment thereof. In some embodiments, the cysteine residue is introduced by mutation in the Fc domain of the antibody, or antigen-binding fragment thereof. For example, the cysteine residue may be selected from the group consisting of Cys118, Cys239, and Cys265. In certain embodiments, the Fc region of the anti-CD45 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 with an EU index as in Kabat. In certain embodiments, the Fc region comprises a D265C mutation. In certain embodiments, the Fc region comprises the D265C and H435A mutations. In certain embodiments, the Fc region comprises the D265C, L234A, and L235A mutations. In certain embodiments, the Fc region comprises the D265C, L234A, L235A, and H435A mutations. In certain embodiments, the Fc region of the anti-CD45 antibody, or antigen-binding fragment thereof, comprises an amino acid substitution at amino acid 239 with an EU index as in Kabat. In certain embodiments, the Fc region comprises an S239C mutation. In certain embodiments, the Fc region comprises an L234A mutation, an L235A mutation, an S239C mutation and a D265A mutation. In another embodiment, the Fc region comprises S239C and H435A mutations. In another embodiment, the Fc region comprises an L234A mutation, an L235A mutation, and an S239C mutation. In yet another embodiment, the Fc region comprises an H435A mutation, an L234A mutation, an L235A mutation, and an S239C mutation. In yet another embodiment, the Fc region comprises an H435A mutation, an L234A mutation, an L235A mutation, an S239C mutation and a D265A mutation.

In particular, the amino acid positions of Fc refer to the EU numbering index unless otherwise noted.

The disclosures of each of the above publications are incorporated by reference in this application as relating to anti-CD45 antibodies. Antibodies and antigen-binding fragments that may be used in combination with the compositions and methods described in this application include the above antibodies and their antigen-binding fragments, as well as humanized variants of the above non-human antibodies and antigen-binding fragments. , And an antibody or antigen-binding fragment that binds to the same epitope as the above antibody and its antigen-binding fragment (eg, as assessed by a competitive antigen-binding assay).

Methods of manipulating antibodies to incorporate any of the Fc modifications of the present application are well known in the art. These methods include, but are not limited to, site-specific (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis for the prepared DNA molecule encoding the antibody or at least the constant region of the antibody. , And cassette mutagenesis. Site-directed mutagenesis is well known in the art (eg Carter et al., Nucleic Acids Res., 13: 4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA. , 82: 488 (1987)). PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17: 723-733 (1989). Another method for preparing sequence variants (cassette mutagenesis) is based on the technique described in Wells et al., Gene, 34: 315-323 (1985).

Methods for Identifying Antibodies To treat cancer, autoimmune diseases, using methods for high-throughput screening of a library of antibodies or antibody fragments that can bind to CD45 expressed by hematopoietic stem cells. , And useful anti-CD45 antibodies may be identified to condition patients in need of hematopoietic stem cell therapy as described in this application (eg, human patients). Such methods may be used to identify improved antibodies AbA, AbB, and AbC. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. Can be mentioned.

The use of phage displays to isolate antibodies or antigen-binding fragments that bind to biologically relevant molecules can be described, for example, in Felici et al., Biotechnol. Annual Rev. 1: 149-183, 1995. The disclosure of each of these, which has been reviewed in Katz, Annual Rev. Biophys. Biomol. Struct. 26: 27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, is in. Incorporated by reference in this application as relating to in vitro display technology. A randomized combinatorial peptide library was constructed as described in Kay, Perspect. Drug Discovery Des. 2: 251-268, 1995 and Kay et al., Mol. Divers. 1: 139-140, 1996. Also, the selection of polypeptides that bind to cell surface antigens has been performed, each of these disclosures being incorporated by reference in this application as being related to the discovery of antigen-binding molecules. Phage display of proteins (eg, multimeric proteins) as functional molecules has been successfully performed (eg, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. See 10: 80-84 1992, the disclosure of each of these is incorporated by reference in this application as relating to the use of in vitro display technology to discover antigen-binding molecules). In addition, functional antibody fragments (eg, Fab and scFv fragments) have also been expressed in in vitro display formats (eg, McCafferty et al., Nature 348: 552-554, 1990; Barbas). See et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982, 1991; and Clackson et al., Nature 352: 624-628, 1991, each of these disclosures discovering an antigen-binding molecule. Incorporated by reference in this application as relating to an in vitro display platform for use). Human anti-CD45 antibodies may also be produced, for example, under the HuMAb-mouse ^{registered trademark} or XenoMouse ^TM . Among other things, these techniques can be used to bind to CD45 expressed by hematopoietic stem cells, and then to reduce endogenous hematopoietic stem cells in patients in need of hematopoietic stem cell transplantation therapy (eg, human patients). Can be used to identify and improve the affinity of antibodies, antibodies, or fragments.

In addition to in vitro display techniques, computational modeling techniques may be used to design and identify antibodies capable of binding to antigens expressed by hematopoietic stem cells (eg, CD45). For example, using computational modeling techniques, one of ordinary skill in the art may bind to a specific epitope (eg, an extracellular epitope of said antigen) on an antigen expressed by a hematopoietic stem cell (eg, CD45). A library of antibodies or antibody fragments may be screened in silico for possible molecules.

Further techniques can be used to identify antibodies or antibody fragments that can bind to CD45 expressed by hematopoietic stem cells and that the cells can incorporate into, for example, by receptor-mediated endocytosis. Sometimes. For example, the in vitro display technique described above may be adapted to screen for an antibody, or antibody fragment thereof, that binds to and is subsequently incorporated into a CD45. Phage displays represent one of the techniques that may be used in combination with this screening paradigm. To identify anti-CD45 antibodies or antibody fragments that may be incorporated into hematopoietic stem cells, one of ordinary skill in the art would describe the phages described in Williams et al., Leukemia 19: 1432-1438, 2005. Display technology may be used (the disclosure of which is incorporated by reference in its entirety in this application). For example, antibodies, antibody fragments (eg, scFv fragments, Fab fragments, diabodies, triabodies, and ¹⁰ Fn3 domains, among others), or ligands (these are randomized) using mutagenesis methods known in the art. Recombinant phage libraries encoding amino acid cassettes [eg, in CDRs or their equivalent regions, or in one or more or all of antibodies or antibody fragments] can be produced. In humans, for example, the skeletal region, hinge, Fc domain, and other regions of the antibody or antibody fragment may have a slightly different sequence compared to, for example, a human germline antibody sequence or a human germline antibody. May be designed to be non-immunogenic.

A phage library containing randomized antibodies or antibody fragments co-bound to phage particles is incubated with CD45 using phage display techniques described in this application or known in the art (eg,). First of all, the phage to remove an antibody exhibiting non-specific protein binding, or a phage encoding an antibody fragment, and to remove an antibody binding to the Fc domain or a phage encoding a fragment thereof. Incubate the library with a blocking agent [eg, milk protein, bovine serum albumin, and / or IgG], and then the phage library with a population of cells (eg, hematopoietic stem cells) expressing CD45. Incubate). Sufficient time for the anti-CD45 antibody, or antibody fragment, to bind to the allogeneic antigen on the cell surface and subsequently be taken up by the hematopoietic stem cells (eg, 30 minutes to 6 hours at 4 ° C., eg, eg. The phage library may be incubated with the hematopoietic stem cells (eg, at 4 ° C. for 1 hour). Subsequently, for the purpose of binding to hematopoietic stem cells and being taken up by hematopoietic stem cells, for example by washing with a cold (4 ° C.) pH 2.8 0.1 M glycine buffer, the CD45. Phage containing antibodies or antibody fragments that do not show sufficient affinity may be removed. Phage bound to an antibody or antibody fragment thereof taken up by the hematopoietic stem cell can be identified, for example, by lysing the cell and recovering the taken up phage from the cell culture medium. be. The phage may then be amplified in the bacterial cells, for example, by incubating the bacterial cells with the recovered phage in 2 × YT medium using methods known in the art. Phage recovered from this medium may then be identified, for example, by determining the nucleic acid sequence of the gene encoding the antibody or antibody fragment inserted into the phage genome. The encoded antibody or antibody fragment is subsequently recombined by chemical synthesis (eg, for the antibody fragment, eg, scFv fragment) or by recombinant expression (eg, for a full-length antibody). May be prepared in.

In addition, the epitope region described in this application is used as peptides, thereby obtaining additional anti-hCD45 antibodies while using the screening techniques disclosed in this application and known in the art. May be screened.

Methods of Production Antibodies may be produced using recombinant methods and compositions, eg, as described in US Pat. No. 4,816,567. In certain embodiments, an isolated nucleic acid encoding the anti-CD45 antibody described in this application is provided. Such nucleic acids may encode an amino acid sequence containing VL of the antibody and / or an amino acid sequence containing VH of the antibody (eg, light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors containing such nucleic acids (eg, expression vectors) are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (eg, transformed below): (1) a nucleic acid encoding an amino acid sequence comprising VL of the antibody and an amino acid sequence comprising VH of the antibody. The vector containing the above, or (2) the first vector containing the nucleic acid encoding the amino acid sequence containing the VL of the present antibody, and the second vector containing the nucleic acid encoding the amino acid sequence containing the VH of the present antibody. In certain embodiments, the host cell is a eukaryote, eg, a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp20 cell). In certain embodiments, a method of making an anti-CLL-1 antibody is provided, wherein a host cell containing a nucleic acid encoding an antibody as provided above is suitable for expression of the antibody. It includes culturing under the above conditions and optionally recovering the antibody from the host cell (or host cell culture medium).

To recombinantly produce an anti-CD45 antibody, a nucleic acid encoding the antibody (eg, a nucleic acid as described above) is isolated, and further cloned and / or expressed in a host cell 1 Insert into more than a seed vector. Such nucleic acids are readily isolated using conventional procedures (eg, by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody). And can be sequenced. Nucleic acids disclosed in this application that may be used to express an antibody or fragment include the nucleic acids set forth in SEQ ID NOs: 25-30 in Table 3.

Suitable host cells for cloning or expressing the vector encoding the antibody include prokaryotic or eukaryotic cells described in this application. For example, antibodies may be produced in bacteria, especially if glycosylation and Fc effector functions are not required. See, for example, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expressing antibody fragments and polypeptides in bacteria (Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana). See also Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated from the bacterial cell paste as a soluble fraction and further purified.

Vertebrate cells can also be used as the host. For example, a mammalian cell line adapted to grow in suspension may be useful. Another example of a useful mammalian host cell line is the SV40-transformed monkey kidney CV1 line (COS-7); a human embryonic kidney cell line (eg, Graham et al., J. Gen Virol. 36:59). (293 or 293 cells as described in (1977)); Baby hamster kidney cells (BHK); Mouse Sertri cells (eg, Mather, Biol. Reprod. 23: 243-251 (1980)). TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); Human lung cells (W138); human liver cells (Hep G2); mouse breast cancer (MMT 060562); TRI cells (eg, Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982). ); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)) and the like. ); And myeloma cell lines (eg, Y0, NS0 and Sp2 / 0, etc.). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C.Lo, ed., Humana Press, Totowa, N.J.), pp. 255. See -268 (2003). In certain embodiments, the host cell is a eukaryote, eg, a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp20 cell).

Allowance for uptake of anti-CD45 antibody, or antibody fragment, may be assessed using, for example, a radionuclide internal uptake assay known in the art. For example, the anti-CD45 antibody or antibody fragment described in this application or identified using in vitro display techniques known in the art can be subjected to radioisotopes (eg, ¹⁸ F, ⁷⁵ Br, ⁷⁷ Br, ¹²² ). I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I, ²¹¹ At, ⁶⁷ Ga, ¹¹¹ In, ⁹⁹ Tc, ¹⁶⁹ Yb, ¹⁸⁶ Re, ⁶⁴ Cu, ⁶⁷ Cu, ¹⁷⁷ Lu, ⁷⁷ As, ⁷² As, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ²¹² Bi, ²¹³ Bi, or ²²⁵ Ac) may be incorporated to make it functional. For example, radioactive halogen (eg, ¹⁸ F, ⁷⁵ Br, ⁷⁷ Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I, ²¹¹ At, etc.) and beads containing an electrophilic halogen reagent (eg, 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, etc.) , Polystyrene beads) (eg, iodinated beads, Thermo Fisher Scientific, Inc., Cambridge, MA) may be used to integrate into the antibody, or antibody fragment. Radiolabeled antibodies, fragments or ADCs thereof may be incubated with hematopoietic stem cells for sufficient time to be incorporated into them (eg, 30 minutes to 6 hours at 4 ° C, 1 hour at 4 ° C, etc.). .. The cells are then washed (eg, using cold (4 ° C.) pH 2.8 0.1 M glycine buffer) to remove antibodies or fragments thereof that have not been incorporated therein. The antibody or antibody fragment taken up therein is compared with the radiation emitted from the obtained hematopoietic stem cells (eg, γ-rays) and the radiation emitted from the recovered wash buffer (eg, γ-rays). May be identified by. The internal uptake assay described above may also be used to characterize the ADC.

Antibody drug conjugate (ADC)
The anti-CD45 antibody described in this application, or an antigen-binding fragment thereof, may be conjugated to a cytotoxin via a linker. In some embodiments, the cytotoxic molecule is conjugated to an antibody or antigen-binding fragment thereof that is incorporated into the cell as disclosed in the present application, and the antibody or fragment thereof is incorporated into the cell. Later, the cytotoxin may approach its intracellular target and mediate the death of hematopoietic cells.

Cytotoxicity Various cytotoxins can be conjugated to an anti-CD45 antibody via a linker for use in the therapies described in this application. In particular, the anti-CD45 ADC comprises an anti-CD45 antibody (or antigen-binding fragment thereof) conjugated (ie, covalently linked by a linker) to a cytotoxic moiety (or cytotoxin). In various embodiments, the cytotoxic moiety is reduced or does not show cytotoxicity when bound as a conjugate. However, after cleavage from the linker, it becomes cytotoxic again. In various embodiments, the cytotoxic moiety retains cytotoxicity without being cleaved from the linker. In some embodiments, the cytotoxic molecule is conjugated to an antibody incorporated into a cell, or an antigen-binding fragment thereof, as described in this application, so that the antibody, or fragment thereof, is a cell. After being incorporated into, the cytotoxin may access its intracellular target and mediate, for example, T cell death.

Thus, the ADC of the invention may be of the general formula Ab- (ZLD) _n , where the antibody or antigen binding fragment (Ab) thereof is conjugated (covalently) to the linker (L) and chemically. Conjugates to cytotoxic moieties (“drugs”, D) through substructures (Z).

Thus, the anti-CD45 antibody or antigen-binding fragment thereof may conjugate to multiple drug moieties as indicated by the integer n, where the integer n represents the average number of cytotoxins per antibody. Here, the integer n may be in the range of, for example, about 1 to about 20. In some embodiments, n is 1-4. In some embodiments, n is 1. The average number of drug moieties per antibody in ADC preparations by conjugation reaction can be determined by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of ADC with respect to n can also be determined. In some cases, by means such as reverse phase HPLC or electrophoresis, homogeneous ADCs with a particular number n can be separated, purified, and characterized from ADCs with other drug loadings. can.

For some anti-CD45 ADCs, n may be limited by the number of binding sites on the antibody. For example, if the bond is a thiol of cysteine, the antibody has only one or several cysteine thiol groups, or has only one or several thiol groups that are reactive enough for the linker to bind. .. In general, antibodies do not contain many free and reactive cysteine groups that can bind to drug moieties; predominantly, the thiol residues of cysteine in the antibody are present as disulfide crosslinks. In certain embodiments, the antibody is reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to produce a thiol group of reactive cysteine under partial or total reducing conditions. May cause you to. In certain embodiments, higher drug loading (DAR) (eg, n> 5) causes agglomeration, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. It can be triggered.

In certain embodiments, less than the theoretical maximum drug moieties are conjugated to the antibody during the conjugation reaction. Antibodies may contain, for example, a drug-linker intermediate or a lysine residue that does not react with a linker reagent, as discussed below. Only the most reactive lysine group can react with the amine-reactive linker reagent. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine to the surface.

The loading of the ADC (drug / antibody ratio) is carried out in various ways (eg, (i) limiting the molar excess of the drug-linker intermediate or linker reagent to the antibody, (ii) the time or temperature of the conjugation reaction. (Iii) Partial or restrictive reducing conditions for thiol modification of cysteine, (iv) cysteine residue to control the number and / or position of linker-drug binding. It may be controlled (by manipulating the amino acid sequence of the antibody by recombinant techniques) to alter the number and position of the groups.

Cell toxins suitable for use with the compositions and methods described in this application include destroying DNA-intercalating agents (eg, anthracyclins), spindle devices known in the art, among others. Can disrupt drugs capable of (eg, binca alkaloids, maytancin, maytancinoids, and derivatives thereof), RNA polymerase inhibitors (eg, amatoxins such as α-amanitin, and derivatives thereof), and protein biosynthesis. Examples include agents (eg, agents exhibiting rRNA N-glycosidase activity such as saporin and lysine A chain).

In some embodiments, the cytotoxin is a microtubule binding agent (eg, maytancin or maytancinoid), amatoxin, pseudomonas exotoxin A, de bouganine, diphtheria toxin, saporin, auristatin, anthracycline, calikeamycin. , Irinotecan, SN-38, Duocarmycin, Pyrrolobenzodiazepine, Pyrrolobenzodiazepine dimer, Indolinobenzodiazepine, Indolinobenzodiazepine dimer, Indolinobenzodiazepine pseudodimer, or variants thereof, or the present application. Other cytotoxic compounds described in or known in the art.

In some embodiments, the antibody-drug conjugate cytotoxin is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the antibody-drug conjugate cytotoxin disclosed in this application is an amatoxin or a derivative thereof (eg, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin). , Amanullinic acid, proamanullin or a derivative thereof, etc.).

Further details regarding cytotoxins that may be used in anti-CD45 ADCs useful in the compositions and methods of the invention are described below.

Amatoxins In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the antibody-drug conjugate cytotoxin disclosed in this application is an amatoxin or a derivative thereof (eg, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin). , Amanullinic acid, proamanullin or a derivative thereof, etc.).

In some embodiments, the antibody-drug conjugate cytotoxin is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof (eg, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, and proamanullin). The structures of various natural amatoxins are disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

ここで、R₁は、H、OH、又はOR_Aである；
R₂は、H、OH、又はOR_Bである；
R_A及びR_Bは、存在する場合、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、又はR_Dである；
R₄は、H、OH、OR_D、又はR_Dである；
R₅は、H、OH、OR_D、又はR_Dである；
R₆は、H、OH、OR_D、又はR_Dである；
R₇は、H、OH、OR_D、又はR_Dである；
R₈は、OH、NH₂、又はOR_Dである；
R₉は、H、OH、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；並びに、
R_Dは、任意選択的に置換されたアルキル（例えば、C₁-C₆アルキル）、任意選択的に置換されたヘテロアルキル（例えば、C₁-C₆ヘテロアルキル）、任意選択的に置換されたアルケニル（例えば、C₂-C₆アルケニル）、任意選択的に置換されたヘテロアルケニル（例えば、C₂-C₆ヘテロアルケニル）、任意選択的に置換されたアルキニル（例えば、C₂-C₆アルキニル）、任意選択的に置換されたヘテロアルキニル（例えば、C₂-C₆ヘテロアルキニル）、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである。 Examples of amatoxins useful in combination with the compositions and methods described in this application include, but are not limited to, compounds according to formula (IV).

Where _{R 1} is H, OH, or ORA;
R ₂ is H, OH, or OR _B ;
_RA and R _B , if present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, or R _D ;
R ₄ is H, OH, OR _D , or R _D ;
R ₅ is H, OH, OR _D , or R _D ;
R ₆ is H, OH, OR _D , or R _D ;
R ₇ is H, OH, OR _D , or R _D ;
R ₈ is OH, NH ₂ , or OR _D ;
R ₉ is H, OH, or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
R _D is optionally substituted alkyl (eg, C ₁ -C ₆ alkyl), optionally substituted heteroalkyl (eg, C ₁ -C ₆ heteroalkyl), optionally substituted. Alkyl (eg, C ₂ -C ₆ alkenyl), optionally substituted heteroalkenyl (eg, C ₂ -C ₆ heteroalkenyl), optionally substituted alkynyl (eg, C ₂ -C ₆ ). _Alkinyl ), optionally substituted heteroalkynyl (eg, C2 _- C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted It is an aryl that has been added, or a heteroaryl that has been optionally substituted.

ここで、R₄、R₅、X、及びR₈は、それぞれ上記で定義されたものである。 For example, in certain embodiments, amatoxins useful in combination with the compositions and methods described in this application include compounds according to formula (IVA).

Here, R ₄ , R ₅ , X, and R ₈ are defined above, respectively.

ここで、R₁は、H、OH、又はOR_Aである；
R₂は、H、OH、又はOR_Bである；
R_A及びR_Bは、存在する場合、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、又はR_Dである；
R₄は、H、OH、OR_D、又はR_Dである；
R₅は、H、OH、OR_D、又はR_Dである；
R₆は、H、OH、OR_D、又はR_Dである；
R₇は、H、OH、OR_D、又はR_Dである；
R₈は、OH、NH₂、又はOR_Dである；
R₉は、H、OH、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；並びに、
R_Dは、任意選択的に置換されたアルキル（例えば、C₁-C₆アルキル）、任意選択的に置換されたヘテロアルキル（例えば、C₁-C₆ヘテロアルキル）、任意選択的に置換されたアルケニル（例えば、C₂-C₆アルケニル）、任意選択的に置換されたヘテロアルケニル（例えば、C₂-C₆ヘテロアルケニル）、任意選択的に置換されたアルキニル（例えば、C₂-C₆アルキニル）、任意選択的に置換されたヘテロアルキニル（例えば、C₂-C₆ヘテロアルキニル）、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである。 For example, in certain embodiments, amatoxins useful in combination with the compositions and methods described in this application include compounds according to formula (IVB):

Where _{R 1} is H, OH, or ORA;
R ₂ is H, OH, or OR _B ;
_RA and R _B , if present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, or R _D ;
R ₄ is H, OH, OR _D , or R _D ;
R ₅ is H, OH, OR _D , or R _D ;
R ₆ is H, OH, OR _D , or R _D ;
R ₇ is H, OH, OR _D , or R _D ;
R ₈ is OH, NH ₂ , or OR _D ;
R ₉ is H, OH, or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
R _D is optionally substituted alkyl (eg, C ₁ -C ₆ alkyl), optionally substituted heteroalkyl (eg, C ₁ -C ₆ heteroalkyl), optionally substituted. Alkyl (eg, C ₂ -C ₆ alkenyl), optionally substituted heteroalkenyl (eg, C ₂ -C ₆ heteroalkenyl), optionally substituted alkynyl (eg, C ₂ -C ₆ ). _Alkinyl ), optionally substituted heteroalkynyl (eg, C2 _- C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted It is an aryl that has been added, or a heteroaryl that has been optionally substituted.

ここで：
R₁は、H、OH、又はOR_Aである；
R₂は、H、OH、又はOR_Bである；
R_A及びR_Bは、存在する場合、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、又はR_Dである；
R₄は、H、OH、OR_D、又はR_Dである；
R₅は、H、OH、OR_D、又はR_Dである；
R₆は、H、OH、OR_D、又はR_Dである；
R₇は、H、OH、OR_D、又はR_Dである；
R₈は、OH、NH₂、又はOR_Dである；
R₉は、H、OH、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；並びに、
R_Dは、任意選択的に置換されたアルキル（例えば、C₁-C₆アルキル）、任意選択的に置換されたヘテロアルキル（例えば、C₁-C₆ヘテロアルキル）、任意選択的に置換されたアルケニル（例えば、C₂-C₆アルケニル）、任意選択的に置換されたヘテロアルケニル（例えば、C₂-C₆ヘテロアルケニル）、任意選択的に置換されたアルキニル（例えば、C₂-C₆アルキニル）、任意選択的に置換されたヘテロアルキニル（例えば、C₂-C₆ヘテロアルキニル）、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである。 In certain embodiments, amatoxins useful in combination with the compositions and methods described in this application include compounds according to formula (IVC):

here:
_{R 1} is H, OH, or ORA;
R ₂ is H, OH, or OR _B ;
_RA and R _B , if present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, or R _D ;
R ₄ is H, OH, OR _D , or R _D ;
R ₅ is H, OH, OR _D , or R _D ;
R ₆ is H, OH, OR _D , or R _D ;
R ₇ is H, OH, OR _D , or R _D ;
R ₈ is OH, NH ₂ , or OR _D ;
R ₉ is H, OH, or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
R _D is optionally substituted alkyl (eg, C ₁ -C ₆ alkyl), optionally substituted heteroalkyl (eg, C ₁ -C ₆ heteroalkyl), optionally substituted. Alkyl (eg, C ₂ -C ₆ alkenyl), optionally substituted heteroalkenyl (eg, C ₂ -C ₆ heteroalkenyl), optionally substituted alkynyl (eg, C ₂ -C ₆ ). _Alkinyl ), optionally substituted heteroalkynyl (eg, C2 _- C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted It is an aryl that has been added, or a heteroaryl that has been optionally substituted.

A synthetic method for producing amatoxin is described in US Pat. No. 9,676,702, which is incorporated by reference in this application.

For example, the antibodies and antigen binding fragments described in this application bind to amatoxin (eg, formula IV, IVA, IVB, or IVC) such that they form a conjugate represented by the formula Ab-Z-L-Am. Where Ab is an antibody, or an antigen-binding fragment thereof, L is a linker, Z is a chemical partial structure, and Am is an amatoxin. Many positions on the amatoxin or derivative thereof can serve as covalent positions with the linking moiety L and, therefore, as covalent positions with the antibody or antigen-binding fragment thereof. be able to.

ここで：
R₁は、H、OH、OR_A、又はOR_Cである；
R₂は、H、OH、OR_B、又はOR_Cである；
R_A及びR_Bは、存在する場合、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、R_C、又はR_Dである；
R₄は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₅は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₆は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₇は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₈は、OH、NH₂、OR_C、OR_D、NHR_C、又はNR_CR_Dである；
R₉は、H、OH、OR_C、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；
R_Cは、-L-Zである；
R_Dは、任意選択的に置換されたアルキル（例えば、C₁-C₆アルキル）、任意選択的に置換されたヘテロアルキル（例えば、C₁-C₆ヘテロアルキル）、任意選択的に置換されたアルケニル（例えば、C₂-C₆アルケニル）、任意選択的に置換されたヘテロアルケニル（例えば、C₂-C₆ヘテロアルケニル）、任意選択的に置換されたアルキニル（例えば、C₂-C₆アルキニル）、任意選択的に置換されたヘテロアルキニル（例えば、C₂-C₆ヘテロアルキニル）、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである；
Lは、任意選択的に置換されたアルキレン（例えば、C₁-C₆アルキレン）、任意選択的に置換されたヘテロアルキレン（例えば、C₁-C₆ヘテロアルキレン）、任意選択的に置換されたアルケニレン（例えば、C₂-C₆アルケニレン）、任意選択的に置換されたヘテロアルケニレン（例えば、C₂-C₆ヘテロアルケニレン）、任意選択的に置換されたアルキニレン（例えば、C₂-C₆アルキニレン）、任意選択的に置換されたヘテロアルキニレン（例えば、C₂-C₆ヘテロアルキニレン）、任意選択的に置換されたシクロアルキレン、任意選択的に置換されたヘテロシクロアルキレン、任意選択的に置換されたアリーレン、任意選択的に置換されたヘテロアリーレン、ペプチド、ジペプチド、-C(=O)-、ジスルフィド、ヒドラゾン、又はそれらの組合せであるような、リンカーである；並びに、
Zは、L上に存在する反応性置換基Z'と、ターゲット抗原（例えば、CD45）に結合する抗体、又はその抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される化学的な部分構造である。 In some embodiments, the amatoxin-linker conjugate Am-LZ is represented by the formula (I).

here:
R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
_RA and R _B , if present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, R _C , or R _D ;
R ₄ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₅ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₆ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₇ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₈ is OH, NH ₂ , OR _C , OR _D , NHR _C , or NR _C R _D ;
R ₉ is H, OH, OR _C , or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
_RC is -LZ;
R _D is optionally substituted alkyl (eg, C ₁ -C ₆ alkyl), optionally substituted heteroalkyl (eg, C ₁ -C ₆ heteroalkyl), optionally substituted. Alkyl (eg, C ₂ -C ₆ alkenyl), optionally substituted heteroalkenyl (eg, C ₂ -C ₆ heteroalkenyl), optionally substituted alkynyl (eg, C ₂ -C ₆ ). _Alkinyl ), optionally substituted heteroalkynyl (eg, C2 _- C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted It is an aryl, or an optionally substituted heteroaryl;
L was optionally substituted alkylene (eg, C ₁ -C ₆ alkylene), optionally substituted heteroalkylene (eg, C ₁ -C ₆ heteroalkylene), optionally substituted. Alkenirene (eg, C ₂ -C ₆ alkenylene), optionally substituted hetero-alkenylene (eg, C ₂ -C ₆ hetero-alkenilen), optionally substituted alkynylene (eg, C ₂ -C ₆ alkinylene) ), Optionally substituted heteroalkynylene (eg, C2 _- C6 _{heteroalkynylene} ), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted. A linker, such as substituted arylene, optionally substituted heteroarylene, peptide, dipeptide, -C (= O)-, disulfide, hydrazone, or a combination thereof;
Z is formed from the coupling reaction between the reactive substituent Z'existing on L and the reactive substituent present in the antibody that binds to the target antigen (eg, CD45) or its antigen-binding fragment. It is a chemical partial structure to be used.

In some embodiments, Am comprises just one _RC substituent.

ここで、Sは、ターゲット抗原に結合する抗体、又はその抗原結合フラグメント内に存在する反応性置換基を表す硫黄原子である(例えば、システイン残基の-SH基に由来する)。 In some embodiments, the LZ

Here, S is a sulfur atom representing an antibody that binds to a target antigen or a reactive substituent present in the antigen-binding fragment thereof (for example, derived from the -SH group of a cysteine residue).

ここで、XはS、SO又はSO₂である、及びAbはAbに結合する位置を示すように示されている。 In some embodiments, the conjugate Am-LZ-Ab is represented by one of formulas III, IIIA, or IIIB:

Here, X is S, SO or SO ₂ , and Ab is shown to indicate the position that binds to Ab.

である、ここで、Abは、Abに結合する位置を示すように示されている。 In some embodiments, Am-LZ-Ab

Here, Ab is shown to indicate a position that binds to Ab.

である、ここで、Abは、Abに結合する位置を示すように示されている。 In some embodiments, Am-LZ-Ab

Here, Ab is shown to indicate a position that binds to Ab.

である、ここで、Abは、Abに結合する位置を示すように示されている。 In some embodiments, Am-LZ-Ab

Here, Ab is shown to indicate a position that binds to Ab.

である、ここで、そのマレイミドが前記抗体中のシステインにあるチオール基と反応する。 In some embodiments, the Am-LZ-Ab precursor, Am-L-Z', is

Here, the maleimide reacts with the thiol group in cysteine in the antibody.

である、ここで、そのマレイミドが前記抗体中のシステインにあるチオール基と反応する。 In some embodiments, the Am-LZ-Ab precursor, Am-L-Z', is

Here, the maleimide reacts with the thiol group in cysteine in the antibody.

ここで：
R₁は、H、OH、OR_A、又はOR_Cである；
R₂は、H、OH、OR_B、又はOR_Cである；
R_A及びR_Bは、存在する場合、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、R_C、又はR_Dである；
R₄は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₅は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₆は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₇は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₈は、OH、NH₂、OR_C、OR_D、NHR_C、又はNR_CR_Dである；
R₉は、H、OH、OR_C、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；
R_Cは、-L-Zである；
R_Dは、任意選択的に置換されたアルキル（例えば、C₁-C₆アルキル）、任意選択的に置換されたヘテロアルキル（例えば、C₁-C₆ヘテロアルキル）、任意選択的に置換されたアルケニル（例えば、C₂-C₆アルケニル）、任意選択的に置換されたヘテロアルケニル（例えば、C₂-C₆ヘテロアルケニル）、任意選択的に置換されたアルキニル（例えば、C₂-C₆アルキニル）、任意選択的に置換されたヘテロアルキニル（例えば、C₂-C₆ヘテロアルキニル）、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである；
Lは、任意選択的に置換されたアルキレン（例えば、C₁-C₆アルキレン）、任意選択的に置換されたヘテロアルキレン（例えば、C₁-C₆ヘテロアルキレン）、任意選択的に置換されたアルケニレン（例えば、C₂-C₆アルケニレン）、任意選択的に置換されたヘテロアルケニレン（例えば、C₂-C₆ヘテロアルケニレン）、任意選択的に置換されたアルキニレン（例えば、C₂-C₆アルキニレン）、任意選択的に置換されたヘテロアルキニレン（例えば、C₂-C₆ヘテロアルキニレン）、任意選択的に置換されたシクロアルキレン、任意選択的に置換されたヘテロシクロアルキレン、任意選択的に置換されたアリーレン、任意選択的に置換されたヘテロアリーレン、ペプチド、ジペプチド、-C(=O)-、ジスルフィド、ヒドラゾン、又はそれらの組合せであるような、リンカーである；
Zは、L上に存在する反応性置換基Z'と、CD45に結合する抗体、又はその抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される化学的な部分構造である；並びに、
ここで、Amは、丁度1個のR_C置換基を含む。 In some embodiments, Am-LZ is represented by equation (IA),

here:
R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
_RA and R _B , if present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, R _C , or R _D ;
R ₄ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₅ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₆ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₇ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₈ is OH, NH ₂ , OR _C , OR _D , NHR _C , or NR _C R _D ;
R ₉ is H, OH, OR _C , or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
_RC is -LZ;
R _D is optionally substituted alkyl (eg, C ₁ -C ₆ alkyl), optionally substituted heteroalkyl (eg, C ₁ -C ₆ heteroalkyl), optionally substituted. Alkyl (eg, C ₂ -C ₆ alkenyl), optionally substituted heteroalkenyl (eg, C ₂ -C ₆ heteroalkenyl), optionally substituted alkynyl (eg, C ₂ -C ₆ ). _Alkinyl ), optionally substituted heteroalkynyl (eg, C2 _- C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted It is an aryl, or an optionally substituted heteroaryl;
L was optionally substituted alkylene (eg, C ₁ -C ₆ alkylene), optionally substituted heteroalkylene (eg, C ₁ -C ₆ heteroalkylene), optionally substituted. Alkenirene (eg, C ₂ -C ₆ alkenylene), optionally substituted hetero-alkenylene (eg, C ₂ -C ₆ hetero-alkenilen), optionally substituted alkynylene (eg, C ₂ -C ₆ alkinylene) ), Optionally substituted heteroalkynylene (eg, C2 _- C6 _{heteroalkynylene} ), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted. A linker, such as substituted arylene, optionally substituted heteroarylene, peptide, dipeptide, -C (= O)-, disulfide, hydrazone, or a combination thereof;
Z is a chemical moiety formed from the coupling reaction between the reactive substituent Z'existing on L and the reactive substituent present in the antibody that binds to CD45 or its antigen-binding fragment. Structure; as well,
Here, Am contains exactly one _RC substituent.

である。 In some embodiments, the LZ

Is.

ここで：
R₁は、H、OH、OR_A、又はOR_Cである；
R₂は、H、OH、OR_B、又はOR_Cである；
R_A及びR_Bは、存在する場合、それらが結合している酸素原子と一緒になって、任意選択的に置換された5員ヘテロシクロアルキル基を形成する；
R₃は、H、R_C、又はR_Dである；
R₄は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₅は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₆は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₇は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₈は、OH、NH₂、OR_C、OR_D、NHR_C、又はNR_CR_Dである；
R₉は、H、OH、OR_C、又はOR_Dである；
Xは、-S-、-S(O)-、又は-SO₂-である；
R_Cは、-L-Zである；
R_Dは、任意選択的に置換されたアルキル（例えば、C₁-C₆アルキル）、任意選択的に置換されたヘテロアルキル（例えば、C₁-C₆ヘテロアルキル）、任意選択的に置換されたアルケニル（例えば、C₂-C₆アルケニル）、任意選択的に置換されたヘテロアルケニル（例えば、C₂-C₆ヘテロアルケニル）、任意選択的に置換されたアルキニル（例えば、C₂-C₆アルキニル）、任意選択的に置換されたヘテロアルキニル（例えば、C₂-C₆ヘテロアルキニル）、任意選択的に置換されたシクロアルキル、任意選択的に置換されたヘテロシクロアルキル、任意選択的に置換されたアリール、又は任意選択的に置換されたヘテロアリールである；
Lは、任意選択的に置換されたアルキレン（例えば、C₁-C₆アルキレン）、任意選択的に置換されたヘテロアルキレン（例えば、C₁-C₆ヘテロアルキレン）、任意選択的に置換されたアルケニレン（例えば、C₂-C₆アルケニレン）、任意選択的に置換されたヘテロアルケニレン（例えば、C₂-C₆ヘテロアルケニレン）、任意選択的に置換されたアルキニレン（例えば、C₂-C₆アルキニレン）、任意選択的に置換されたヘテロアルキニレン（例えば、C₂-C₆ヘテロアルキニレン）、任意選択的に置換されたシクロアルキレン、任意選択的に置換されたヘテロシクロアルキレン、任意選択的に置換されたアリーレン、任意選択的に置換されたヘテロアリーレン、ペプチド、ジペプチド、-C(=O)-、ジスルフィド、ヒドラゾン、又はそれらの組合せであるような、リンカーである；
Zは、L上に存在する反応性置換基と、CD45に結合する抗体、又はその抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される化学的な部分構造である；並びに、
ここで、Amは、丁度1個のR_C置換基を含む。 In some embodiments, Am-LZ is represented by equation (IB),

here:
R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
_RA and R _B , if present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, R _C , or R _D ;
R ₄ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₅ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₆ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₇ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₈ is OH, NH ₂ , OR _C , OR _D , NHR _C , or NR _C R _D ;
R ₉ is H, OH, OR _C , or OR _D ;
X is -S-, -S (O)-, or -SO _2- ;
_RC is -LZ;
R _D is optionally substituted alkyl (eg, C ₁ -C ₆ alkyl), optionally substituted heteroalkyl (eg, C ₁ -C ₆ heteroalkyl), optionally substituted. Alkyl (eg, C ₂ -C ₆ alkenyl), optionally substituted heteroalkenyl (eg, C ₂ -C ₆ heteroalkenyl), optionally substituted alkynyl (eg, C ₂ -C ₆ ). _Alkinyl ), optionally substituted heteroalkynyl (eg, C2 _- C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted Alyl, or optionally substituted heteroaryl;
L was optionally substituted alkylene (eg, C ₁ -C ₆ alkylene), optionally substituted heteroalkylene (eg, C ₁ -C ₆ heteroalkylene), optionally substituted. Alkenirene (eg, C ₂ -C ₆ alkenylene), optionally substituted hetero-alkenylene (eg, C ₂ -C ₆ hetero-alkenilen), optionally substituted alkynylene (eg, C ₂ -C ₆ alkinylene) ), Optionally substituted heteroalkynylene (eg, C2 _- C6 _{heteroalkynylene} ), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted. A linker, such as substituted arylene, optionally substituted heteroarylene, peptide, dipeptide, -C (= O)-, disulfide, hydrazone, or a combination thereof;
Z is a chemical partial structure formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody that binds to CD45 or an antigen-binding fragment thereof. Yes; and
Here, Am contains exactly one _RC substituent.

である。 In some embodiments, the LZ

Is.

ここで、Yは-C(=O)-、-C(=S)-、-C(=NR_E)-, 又は-C(R_ER_E’)-である；並びに、
R_E及びR_E'は、それぞれ独立して、任意選択的に置換されたC₁-C₆アルキレン-R_C、任意選択的に置換されたC₁-C₆ヘテロアルキレン-R_C、任意選択的に置換されたC₂-C₆アルケニレン-R_C、任意選択的に置換されたC₂-C₆ヘテロアルケニレン-R_C、任意選択的に置換されたC₂-C₆アルキニレン-R_C、任意選択的に置換されたC₂-C₆ヘテロアルキニレン-R_C、任意選択的に置換されたシクロアルキレン-R_C、任意選択的に置換されたヘテロシクロアルキレン-R_C、任意選択的に置換されたアリーレン-R_C、又は任意選択的に置換されたヘテロアリーレン-R_C、である。 In some embodiments, _RA and R _B , if present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the following equation:

Where Y is -C (= O)-, -C (= S)-, -C (= N R _E )-, or -C (R _E R _E' )-;
R _E and R _E'are independent and optionally substituted C ₁ -C ₆ alkylene-R _C , optionally substituted C ₁ -C ₆ heteroalkylene-R _C , optional. C ₂ -C ₆ alkenylene-R _C , optionally substituted C ₂ -C ₆ heteroalkenilen-R _C , optionally substituted C ₂ -C ₆ alkinylene-R _C , Optionally substituted C ₂ -C ₆ heteroalkynylene-R _C , optionally substituted cycloalkylene-R _C , optionally substituted heterocycloalkylene-R _C, optionally substituted Substituted arylene- _RC or optionally substituted heteroarylen- _RC .

R₃は、H、又はR_Cである；
R₄は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₅は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₆は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₇は、H、OH、OR_C、OR_D、R_C、又はR_Dである；
R₈は、OH、NH₂、OR_C、又はNHR_Cである；
R₉は、H、又はOHである；
Xは、 -S-、-S(O)-、又は-SO₂-である；並びに、
ここで、R_C及びR_Dは、それぞれ上記に規定した通りである。 In some embodiments, Am-LZ is represented by Equation (IA) or Equation (IB).
Where R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
R _A and R _B , if present, together with the oxygen atoms to which they are attached form:

R ₃ is H, or R _C ;
R ₄ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₅ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₆ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₇ is H, OH, OR _C , OR _D , _RC , or R _D ;
R ₈ is OH, NH ₂ , OR _C , or NHR _C ;
R ₉ is H, or OH;
X is -S-, -S (O)-, or -SO _2- ;
Here, R _C and R _D are as specified above, respectively.

R₃は、H、又はR_Cである；
R₄及びR₅は、それぞれ独立してH、OH、OR_C、R_C、又はOR_Dである；
R₆及びR₇は、それぞれHである；
R₈は、OH、NH₂、OR_C、又はNHR_Cである；
R₉は、H、又はOHである；
Xは、 -S-、-S(O)-、又は-SO₂-である；並びに、
ここで、R_Cは、上記に規定した通りである。 In some embodiments, Am-LZ is represented by Equation (IA) or Equation (IB).
Where R ₁ is H, OH, _{ORA, or OR C ;}
R ₂ is H, OH, OR _B , or OR _C ;
R _A and R _B , if present, together with the oxygen atoms to which they are attached form:

R ₃ is H, or R _C ;
R ₄ and R ₅ are independently H, OH, OR _C , _RC , or OR _D ;
R ₆ and R ₇ are H, respectively;
R ₈ is OH, NH ₂ , OR _C , or NHR _C ;
R ₉ is H, or OH;
X is -S-, -S (O)-, or -SO _2- ;
Here, R _C is as specified above.

R₃、R₄、R₆、及びR₇は、それぞれHである；
R₅は、OR_Cである；
R₈は、OH、又はNH₂である；
R₉は、H、又はOHである；
Xは、 -S-、-S(O)-、又は-SO₂-である；並びに、
ここで、R_Cは、上記に規定した通りである。このようなアマトキシン・コンジュゲートは、例えば、米国特許出願公開第2016/0002298号に記載されており、その開示は、その全体が本出願に参照により取り込まれる。 In some embodiments, Am-LZ is represented by Equation (IA) or Equation (IB).
Where _{R 1} is H, OH, or ORA;
R ₂ is H, OH, or OR _B ;
R _A and R _B , if present, together with the oxygen atoms to which they are attached form:

R ₃ , R ₄ , R ₆ , and R ₇ are H, respectively;
R ₅ is OR _C ;
R ₈ is OH, or NH ₂ ;
R ₉ is H, or OH;
X is -S-, -S (O)-, or -SO _2- ;
Here, R _C is as specified above. Such amatoxin conjugates are described, for example, in US Patent Application Publication No. 2016/0002298, the disclosure of which is incorporated by reference in its entirety in this application.

In some embodiments, Am-LZ is represented by Equation (IA) or Equation (IB).
Where R ₁ and R ₂ are H or OH independently, respectively;
R ₃ is R _C ;
R ₄ , R ₆ , and R ₇ are H, respectively;
R ₅ is H, OH, or OC ₁ -C ₆ alkyl;
R ₈ is OH, or NH ₂ ;
R ₉ is H, or OH;
X is -S-, -S (O)-, or -SO _2- ;
Here, R _C is as specified above. Such amatoxin conjugates are described, for example, in US Patent Application Publication No. 2014/0294865, the disclosure of which is incorporated by reference in its entirety in this application.

In some embodiments, Am-LZ is represented by Equation (IA) or Equation (IB).
Where R ₁ and R ₂ are H or OH independently, respectively;
R ₃ , R ₆ , and R ₇ are H, respectively;
R ₄ and R ₅ are independently H, OH, OR _C , or _RC ;
R ₈ is OH, or NH ₂ ;
R ₉ is H, or OH;
X is -S-, -S (O)-, or -SO _2- ;
Here, R _C is as specified above. Such amatoxin conjugates are described, for example, in US Patent Application Publication No. 2015/0218220, the disclosure of which is incorporated by reference in its entirety in this application.

In some embodiments, Am-LZ is represented by Equation (IA) or Equation (IB).
here:
R ₁ and R ₂ are independently H or OH;
R ₃ , R ₆ , and R ₇ are H, respectively;
R ₄ and R ₅ are independently H or OH;
R ₈ is OH, NH ₂ , OR _C , or NHR _C ;
R ₉ is H, or OH;
X is -S-, -S (O)-, or -SO _2- ;
Here, R _C is as specified above. Such amatoxin conjugates are described, for example, in US Pat. Nos. 9,233,173 and 9,399,681, and US 2016/0089450, the disclosure of each of which is incorporated herein by reference in its entirety.

である。 In some embodiments, Am-L-Z'is

Is.

Further amatoxins that can be used for conjugation to an antibody or antigen-binding fragment thereof according to the compositions and methods described in this application are, for example, WO 2016/142049; WO 2016/071856; WO 2017/149077; WO. 2018/115466; and WO 2017/046658, the disclosure of each of these is incorporated by reference in its entirety in this application.

ここで、Xは、S、SO、又はSO₂である；R₁は、H又は化学的な部分構造Zを介して前記抗体若しくはその抗原結合フラグメントに共有結合したリンカーであり、Zは、前記リンカー上に存在する反応性置換基と、抗体若しくはその抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される；並びに、R₂は、H又は化学的な部分構造Zを介して前記抗体若しくはその抗原結合フラグメントに共有結合したリンカーであり、Zは、前記リンカー上に存在する反応性置換基と、抗体若しくはその抗原結合フラグメント内に存在する反応性置換基との間のカップリング反応から形成される；ここで、R₁がHの場合、R₂がリンカーで、R₂がHの場合、R₁がリンカーである。 In some embodiments, Am-LZ is represented by Equation (II), Equation (IIA), or Equation (IIB).

Where X is S, SO, or SO ₂ ; R ₁ is a linker covalently attached to the antibody or its antigen-binding fragment via H or the chemical partial structure Z, where Z is said. It is formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or its antigen-binding fragment; and R ₂ is H or a chemical partial structure. A linker covalently bonded to the antibody or its antigen-binding fragment via Z, wherein Z is a reactive substituent present on the linker and a reactive substituent present in the antibody or its antigen-binding fragment. Formed from the coupling reaction between; where R ₁ is H, then R ₂ is the linker, and R ₂ is H, then R ₁ is the linker.

である。 In some embodiments, R ₁ is a linker, and R ₂ is H, and the linker and chemical partial structure together are as LZ.

Is.

である。 In some embodiments, R ₁ is a linker, and R ₂ is H, and the linker and chemical partial structure together are as LZ.

Is.

である。 In one embodiment, Am-LZ-Ab is:

Is.

である。 In one embodiment, Am-LZ-Ab is:

Is.

である、ここで、そのマレイミドが前記抗体中のシステインにあるチオール基と反応する。 In some embodiments, the Am-LZ-Ab precursor (ie, Am-L-Z') is one of the following:

Here, the maleimide reacts with the thiol group in cysteine in the antibody.

In some embodiments, the cytotoxin is α-amanitin. In some embodiments, the α-amanitin is bound to the anti-CD45 antibody via linker L. In some embodiments, the α-amanitin is a compound of formula IV. The linker L binds to any one of several possible positions of α-amanitin in formula IV (eg, any of R ¹ -R ⁹ ) and formulas I, IA, IB, II, IIA. , Or may provide an α-amanitin-linker conjugate of IIB. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is β-amanitin. In some embodiments, the β-amanitin is bound to the anti-CD45 antibody via linker L. In some embodiments, the β-amanitin is a compound of formula IV. The linker L binds to any one of several possible positions of β-amanitin in formula IV (eg, any of R ¹ -R ⁹ ) and formulas I, IA, IB, II, IIA. , Or may provide a β-amanitin-linker conjugate of IIB. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is γ-amanitin. In some embodiments, the γ-amanitin is bound to the anti-CD45 antibody via linker L. In some embodiments, the γ-amanitin is a compound of formula IV. The linker L binds to any one of several possible positions of γ-amanitin of formula IV (eg, any of R ¹ -R ⁹ ) and formulas I, IA, IB, II, IIA. , Or may provide a γ-amanitin-linker conjugate of IIB. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is ε-amanitin. In some embodiments, the ε-amanitin is bound to the anti-CD45 antibody via linker L. In some embodiments, the ε-amanitin is a compound of formula IV. The linker L binds to any one of several possible positions of ε-amanitin of formula IV (eg, any of R ¹ -R ⁹ ) and formulas I, IA, IB, II, IIA. , Or may provide an ε-amanitin-linker conjugate of IIB. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is amanin. In some embodiments, the amanin is bound to the anti-CD45 antibody via linker L. In some embodiments, the amanin is a compound of formula IV. The linker L binds to any one of several possible positions of amanin in formula IV (eg, any of R ¹ -R ⁹ ) and formula I, IA, IB, II, IIA, or May provide IIB amanin-linker conjugates. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is amaninamide. In some embodiments, the amaninamide is bound to the anti-CD45 antibody via linker L. In some embodiments, the amaninamide is a compound of formula IV. The linker L binds to any one of several possible positions of amaninamide of formula IV (eg, any of R ¹ -R ⁹ ) and formula I, IA, IB, II, IIA, or May provide amaninamide-linker conjugates of IIB. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is amanullin. In some embodiments, the amanullin is bound to the anti-CD45 antibody via linker L. In some embodiments, the amanullin is a compound of formula IV. The linker L binds to any one of several possible positions of amanullin in formula IV (eg, any of R ¹ -R ⁹ ) and formula I, IA, IB, II, IIA, or May provide IIB amanullin-linker conjugates. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is amanullinic acid. In some embodiments, the amanullinic acid is bound to the anti-CD45 antibody via linker L. In some embodiments, the amanullinic acid is a compound of formula IV. The linker L binds to any one of several possible positions of amanullinic acid of formula IV (eg, any of R ¹ -R ⁹ ) and formulas I, IA, IB, II, IIA, Alternatively, IIB amanullinic acid-linker conjugates may be provided. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

In some embodiments, the cytotoxin is proamanullin. In some embodiments, the proamanullin is bound to the anti-CD45 antibody via linker L. In some embodiments, the proamanullin is a compound of formula IV. The linker L binds to any one of several possible positions of proamanullin of formula IV (eg, any of R ¹ -R ⁹ ) and formula I, IA, IB, II, IIA, or May provide IIB proamanullin-linker conjugates. In some embodiments, the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a partial PAB-Cit-Val. In some embodiments, the linker comprises a partial PAB-Ala-Val. In some embodiments, the linker comprises a-((C = O) (CH ₂ ) _n -unit, where n is an integer of 1-6.

である。 In some embodiments, the linker comprises a-(CH ₂ ) _n -unit. Where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C = O) (CH ₂ ) _n- . In some embodiments, the linker is -PAB-Ala-Val- ((C = O) (CH 2) n-. (C = O) (CH ₂ ) _n- . In some embodiments, the linker L and the chemical partial structure Z together, as LZ,

Is.

Antibodies and antigen-binding fragments for use with the compositions and methods described in this application may be α-amanitin or variants thereof, using techniques known in the art or conjugated as described in this application. Can be conjugated to the amatoxin of. For example, an antibody that recognizes and binds to a target antigen, and its antigen-binding fragment (anti-CD45 antibody), should be conjugated to an amatoxin such as α-amanitin or a variant thereof, as described in US 2015/0218220. And its disclosure is incorporated by reference in this application as it relates to, for example, amatoxins such as α-amanitin and variants thereof, and covalent linkers that can be used to covalently conjugate. ..

Auristatins The anti-CD45 antibody or antigen-binding fragment thereof described in this application may be conjugated to a cytotoxin that is auristatin (US Pat. No. 5,635,483; 5,780,588). Auristatins are anti-mitotic agents that interfere with microtubule dynamics, GTP hydrolysis, fission and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584), It also has anticancer activity (US Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42: 2961-2965). (US Pat. No. 5,635,483; 5,780,588). The auristatin drug moiety may bind to the antibody via the N- (amino) or C- (carboxyl) terminus of the peptide drug moiety (WO02 / 088172).

Exemplary auristatin embodiments include N-terminally bound monomethyl auristatin drug moieties DE and DF, which are Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, 2004. Published March 28 (this disclosure is incorporated by reference in its entirety in this application).

An exemplary auristatin embodiment is MMAE, where the wavy line is directed to the linker of the antibody-linker conjugate (as described in this application, -LZ-Ab or -L-Z'). Indicates the covalent bond position.

Another exemplary embodiment of auristatin is MMAF, where the wavy line is an antibody-linker conjugate, as disclosed in US 2005/0238649 (as described in this application). Indicates the position to covalently bind to the linker of -LZ-Ab or -L-Z').

Auristatins can be prepared according to the following methods: US Pat. No. 5,635,483; US Pat. No. 5,780,588; Pettitt et al (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al (1998). ) Anti-Cancer Drug Design 13: 243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 15: 859-863; and Doronina (2003) Nat. Biotechnol. 21 (7): 778-784.

Maytansinoids The antibodies described in this application and their antigen-binding fragments may be conjugated to cytotoxins, which are microtubule binders. In some embodiments, the microtubule binder is maitansine, maytansinoid or a maytansinoid analog. Maytansinoids are mitotic inhibitors that act by binding to microtubules and inhibiting tubulin polymerization. Maitansine was first isolated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytancinoids (eg, maytansinol and C-3 maytansinol esters) (US Pat. No. 4,151,042). Synthetic Maytansinol, as well as its derivatives and analogs thereof, are described, for example, in US Pat. 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. The drug portion of maytancinoid is an attractive drug portion in antibody drug conjugates. Because the drug portion of the maytansinoid is relatively accessible to prepare by (i) fermentation or chemical modification, derivatization of the fermented product, (ii) antibody via a non-disulfide linker. It is easy to derivatize with a functional group suitable for conjugate to, (iii) stable in plasma, and (iv) effective against various tumor cell lines.

Examples of suitable Maytansinoids include esters of Maytancinol, synthetic Maytansinol, and Maytansinol analogs and derivatives. Included in this application are any cellular toxins, such as maytancinoids, maytancinols, and maytancinol analogs, and derivatives that inhibit microtubule formation and are highly toxic to mammalian cells. ..

Examples of suitable Maytansinol esters include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids are described in US Pat. No. 4,137,230; 4,151,042; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,364,866; 4,424,219; 4,450,254; 4,322,348; 4,362,663; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497; As such, it is incorporated by reference in this application.

。 In some embodiments, the antibody-drug conjugates (ADCs) of the present disclosure, as cytotoxic drugs, are thiol-containing maitansinoids (DM1) (formally N ^{2'-deacetyl-N 2 '} -(3-). Mercapto-1-oxopropyl) -called maitansine) is used. DM1 is represented by the following structural formula V:

..

。 In another embodiment, the conjugates of the invention cell the thiol-containing maytansinoid N 2'-deacetyl-N ² '(4-methyl-4-mercapto- ¹ -oxopentyl) -maitansine (eg DM4). Used as a toxic drug. DM4 is represented by the following structural formula VI:

..

。 Another maitansinoid containing a side chain containing a steric hindrance thiol bond is N 2'-deacetyl-N ^{-2 '} (4-mercapto-1-oxopentyl) -maitansine (called DM3) and has the following structure: Expressed by Equation VII:

..

Each Maytansinoid taught in US Pat. Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the invention. In this regard, the entire disclosures of 5,208,020 and 7,276,697 are incorporated by reference in this application.

Many positions on the matanthinoids can serve as covalent positions with the linking moiety and, therefore, with the antibody or antigen-binding fragment thereof. Yes (-L-Z-Ab or -L-Z'as described in this application). For example, the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy, and the C-20 position with a hydroxy group are all expected to be useful. To. In some embodiments, the C-3 position serves as a covalent position with the linker partial structure, and in some specific embodiments, the C-3 position of Maytancinol is covalently associated with the linking moiety. It serves as a covalent bond position. There are many linking groups known in the art for making antibody-maytansinoid conjugates, for example, they are US Pat. No. 5,208,020, 6,441,163, and European Patent No. 0425235 B1; Chari. Et al., Cancer Research 52: 127-131 (1992); as well as those disclosed in U.S. 2005/0169933 A1 (these disclosures are expressly incorporated by reference in this application). Further linking groups are described and exemplified in this application.

The invention also includes various isomers and mixtures of Maytansinoids and conjugates. Certain compounds and conjugates of the invention may exist in the form of various stereoisomers, enantiomers, and diastereomers. Generating such antibody-maytansinoid conjugates is described in US Pat. Nos. 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, and 7,368,565 (each of which is incorporated in its entirety into this application). ).

Anthracyclines In other embodiments, the antibodies described in this application and their antigen-binding fragments may be conjugated to cytotoxins, which are anthracycline molecules. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Studies have shown that anthracycs can damage cells by a number of different mechanisms, including: 1) by intercalating the drug molecule into the cell's DNA. , Inhibiting DNA-dependent nucleic acid synthesis; 2) Production of free radicals by drugs, free radicals then react with cellular polymers to cause cell damage, or 3) interaction between drug molecules and cell membranes Action [See, for example, C. Peterson et al., "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia" in Anthracycline Antibiotics In Cancer Therapy; NR Bachur, "Free Radical Damage" id. At pp.97-102. thing]. Due to the potential for cytotoxicity, anthracyclines have been used to treat a number of cancers such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma and sarcoma [eg PH- Wiernik, in Anthracycline: Current] . See Status And New Developments p 11]. Commonly used anthracycs include doxorubicin, epirubicin, idarubicin and daunomycin. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. Typical examples of anthracyclines are, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (adriamycin; Bedford Laboratories; hydrochloric acid). Includes doxorubicin, hydroxy-daunorubicin, and also called Rubex), epirubicin (Ellence; Physer), and idalubicin (Idamycin; Physer).

The anthracycline analog, doxorubicin (ADRIAMYCINO), is thought to interact with DNA by intercalation and inhibit the progression of the enzyme topoisomerase II, which unwinds DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after cleaving the DNA strand for replication, preventing the DNA double helix from closing again, thereby stopping the process of replication. Doxorubicin and DAUNOMYCIN are prototype cytotoxic natural anthracycline chemotherapeutic agents (Sessa et al., (2007) Cardiovasc. Toxicol. 7: 75-79).

。 A non-limited example of anthracycline suitable for use in this application is PNU-159682 (“PNU”). PNU is more than 3000-fold more cytotoxic than its parent, nemorphicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU is represented by the following structural formula:

..

Multiple positions on anthracyclines such as PNU serve as covalently bound positions to said linking moieties and, therefore, with anti-CD45 antibodies or antigen-binding fragments thereof as described in this application. It can serve as a covalently bonded position. For example, a linker may be introduced via modification to the hydroxymethyl-ketone side chain.

ここで、波線は、本出願に記載するようなADCのリンカーに共有結合する位置を示す。 In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

Here, the wavy line indicates the position to covalently bond to the linker of the ADC as described in this application.

ここで、波線は、本出願に記載するようなADCのリンカーに共有結合する位置を示す。 In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

Here, the wavy line indicates the position to covalently bond to the linker of the ADC as described in this application.

Pyrrolobenzodiazepines (PBDs) and the like In other embodiments, the anti-CD45 antibody described in this application, or an antigen-binding fragment thereof, is conjugated to a cytotoxin that is pyrolobenzodiazepine (PBD) or to a cytotoxin containing PBD. I have something to do. PBDs are natural products produced by certain actinomycetes and have been shown to be sequence-selective DNA alkylating compounds. PBD cytotoxins include, but are not limited to, anthramycin, dimeric PBDs, and, for example, Hartley, JA (2011). “The development of pyrrolobenzodiazepines as antitumour agents.” Expert Opin. Inv. Drug, 20 (6), 733-744; and Antonow, D .; Thurston, DE (2011) “Synthesis of DNA-interactive pyrrolo [2,1-c] [1,4] benzodiazepines (PBDs).” Chem. Rev. 111: 2815-2864 includes those disclosed.

ここで、波線は、リンカーに結合する位置を示す。 In some embodiments, the cytotoxin is a pyrolobenzodiazepine dimer represented by the following formula:

Here, the wavy line indicates the position to bind to the linker.

In some embodiments, the cytotoxin is conjugated to the antibody, or an antigen-binding fragment thereof, via a maleimide caproyl linker.

In some embodiments, the linker is a peptide, oligosaccharide,-(CH ₂ ) _p -,-(CH ₂ CH ₂ O) _q -,-(C = O) (CH ₂ ) _r -,-(CH 2) r-,-(CH 2) C = O) (CH ₂ CH ₂ O) _t -,-(NHCH ₂ CH ₂ ) _u- , -PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac)-PAB, Phe-Lys-PAB, Phe-Lys (Ac)-PAB, D-Val-Leu-Lys, Gly-Gly -Contains one or more of Arg, Ala-Ala-Asn-PAB, or Ala-PAB, where p, q, r, t, and u are integers from 1 to 12. In each case, it is selected independently.

ここで、R₁は、CH₃(Ala)又は(CH₂)₃NH(CO)NH₂(Cit)、である。 In some embodiments, the linker has the structure of the following formula:

Where R ₁ is CH ₃ (Ala) or (CH ₂ ) ₃ NH (CO) NH ₂ (Cit).

ここで、波線は、前記細胞毒素(例えば、PBD)に結合する位置を示す。ある特定の実施形態では、R₁はCH₃である。 In some embodiments, the linker comprises a reactive substituent Z'before conjugating to the antibody and together, as L-Z', has the following structure:

Here, the wavy line indicates the position of binding to the cytotoxin (eg, PBD). In certain embodiments, R ₁ is CH ₃ .

。 In some embodiments, the cytotoxin-linker conjugate comprises the reactive substituent Z'before conjugating to the antibody, together as Cy-L-Z', given the following structural formula: Have:

..

This particular cytotoxic-linker conjugate is known as tecillin (SG3249) and is described, for example, in Howard et al., ACS Med. Chem. Lett. 2016, 7 (11), 983-987. This disclosure, in its entirety, is incorporated herein by reference.

ここで、波線は、前記リンカーに結合する位置を示す。 In some embodiments, the cytotoxin is a pyrolobenzodiazepine dimer represented by the following formula:

Here, the wavy line indicates a position to bind to the linker.

。 In some embodiments, the cytotoxin-linker conjugate comprises a reactive substituent Z'before conjugating to the antibody and together has the following structure as Cy-L-Z'. :

..

This particular cytotoxin-linker conjugate is known as talylin, for example, in connection with the ADC Vadastuximab talirine (SGN-CD33A), Mantaj et al., Angewandte Chemie International Edition English 2017, This disclosure, described in 56, 462-488, is incorporated herein by reference in its entirety.

ここで、波線は、前記リンカーに結合する位置を示す。 In some embodiments, the cytotoxin is an indolinobenzodiazepine pseudodimer having the following structural formula:

Here, the wavy line indicates a position to bind to the linker.

これは、ADC IMGN632（例えば、国際特許出願公開第WO2017004026号［これは、本出願に参照により取り込まれる］に開示されている）を含む。 In some embodiments, the cytotoxin-linker conjugate comprises a reactive substituent Z'before conjugating to the antibody and together has the following structure as Cy-L-Z'. :

This includes ADC IMGN632 (eg, disclosed in International Patent Application Publication No. WO2017004026 [which is incorporated by reference in this application]).

Calicaremycin In other embodiments, the antibodies described in this application and antigen-binding fragments thereof may be conjugated to a cytotoxin that is an energyin antitumor antibiotic (eg, Calicaremycins, Ozogamicin). The Calicaremycin family of antibiotics can cleave double-stranded DNA at concentrations below picomols. See US Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all relating to the American Cyanamid Company) for the preparation of conjugates of the Calicaremycin family. Structural analogs of calikeamycin that can be used include, but are not limited to, for example, Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925- 2928 (1998), and the above-mentioned US patents relating to American Cyanamid, which are disclosed.

。 The exemplary Calicaremycin is named γ ₁ , which is simply referred to in this application as gamma and has the following structural formula:

..

In some embodiments, the calikeamycin is a gamma-caricaemycin derivative or an N-acetyl gamma-caricaemycin derivative. Structural analogs of calikeamycin that may be used include, but are not limited to, Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998), and those disclosed in the above US patents. Calicaremycins contain a methyltrisulfide moiety that can react with a suitable thiol to form a disulfide, which at the same time provides a Calicareamycin derivative to the anti-CD45 antibody or anti-CD45 antibody described in this application. A functional group useful for binding via a linker is introduced into the antigen-binding fragment. See US Patents 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all American Cyanamid Company) for preparing conjugates of the Calicaremycin family. Structural analogs of calikeamycin that may be used include, but are not limited to, Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998), and those disclosed in the above US patents for American Cyanamid.

ここで、波線は、前記リンカーに結合する位置を示す。 In certain embodiments, the ADC cytotoxin disclosed in this application is a calikeamycin disulfide derivative represented by the following formula:

Here, the wavy line indicates a position to bind to the linker.

Additional Cell Toxins In other embodiments, the antibodies described in this application and their antigen-binding fragments may be conjugated to cell toxins other than or in addition to the cell toxins disclosed above in this application. .. Additional estrogen suitable for use with the compositions and methods described in this application are, but are not limited to, 5-ethynyluracil, abilaterone, acylfulvene, adecypenol. ), Adzelesin, aldesleukin, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrin Anagrelide, anastrozole, andrographolide, angiopoptosis inhibitor, antarelix, anti-dorsalizing morphogenetic protein-1, anti-androgen , Prostate cancer, anti-estrogen, anti-neoplastic drug (antineoplaston), antisense oligonucleotide, aphidicolin glycinate, apoptotic gene modulator, apoptotic regulator, apurinic acid, asulacrine, Atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III. ) Derivatives, balanol, batimastat, BCR / ABL antagonist, benzochlorin, benzoylstaurosporine, β-lactam derivative, β-alethine, βclamycin B, betaclamycin Acids, bFGF inhibitors, bicalutamide, bisantrene, bisaziri Ginylspermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionin sulfoximine ), Calcipotriol, calphostin C, camptothecin derivative (eg, 10-hydroxy-camptothecin), capecitabine, carboxamide-amino-triazole, carboxamide triazole, carzelesin, Casein kinase inhibitor, castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine ( cladribine), clomiphene and its analogs, clotrimazole, collismycin A, corismycin B, combretastatin A4, combretastatin analog, conagenin, clambecidin (crambescidin) 816, crisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocphosphert cytarabine ocfosfate), cytolytic factor, cytostatin, dacriximab, decitabine, dehydrodidemnin B, 2'deoki Sicoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermin (diethylnorspermine), dihydro-5-azacitidine, dihydrotaxol, dioxamycin, diphenyl spiromustine, discodermolide, docosanol, dolasetron, doxifluridine , Droroxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemen , Emitefur, epothilones, epitilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate (Also known as etopophos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridor, fleze Fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, Fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, geratinase inhibitor, gemcitabine, glutathylone inhibitor , Homoharingtonine (HHT), hypericin, Ibandronic acid, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imikimodo imiquimod), immunostimulatory peptide, iobenguane, iododoxorubicin, ipomeanol, irinotecan, iroplact, irsogladine, isobengazole, jaspraquinolide jas (kahalalide) F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, retrozol ( letrozole, lipophilic platinum compound, lissoclinamide 7, lobaplatin, lometrexol, londamine, losoxantrone, loxoribine, lurtotecan, lurtotecan, lutetium ), Lysofylline, massoprocol, maspin, matrix metalloproteinase inhibitor, menogaril, me Lubarone, meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, mirimostim, mithracin, mitoguazone, mitoguazone mitolactol, mitomycin and its analogs, mitonafide, mitoxantrone, mofarotene, molgramostim, mycapeloxide B, myriaporone, N-acetyldinaline, N-substituted benzamide, nafarelin, nagrestip, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, Nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaliplatin Unomycin (oxaunomycin), paclitaxel and its analogs, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazeliptin pa ), Pegaspargase, peldesine, sodium polysulfate, pentostatin, pentatrozole, perflubron, perphosphami Do (perfosfamide), phenazinomycin, picibanil, pirarubicin, pyritrexim, podophyllotoxin, porfiromycin, purin nucleoside phosphorylase inhibitor, Raltitrexed, rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol, saintopin, saintopin, sarcofitol sargramostim, sobuzoxane, sonermin, sparfosic acid, spicamycin D, spiromustine, spiposide, sulfinosine, talimustine tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsentin, tricilibine, trimetribine, trimetribine ), Vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

Linkers As used in this application, the term "linker" refers to a divalent chemical partial structure that comprises a covalent bond or chain of atoms that is covalently attached to an anti-CD45 antibody-drug conjugate (ADC) of formula I. means. Suitable linkers have two reactive ends, one for conjugating to an antibody and the other for conjugating to a cytotoxin. The reactive end (reactive partial structure, Z') of the linker to be conjugated to the antibody is typically conjugated to the antibody via a cysteine thiol or lysine amine group on the antibody. Therefore, a group that is reactive with thiol, such as a double bond (eg, maleimide), or a desorbing group such as chloro, bromo, iodine, or an R-sulfanyl group. , Or a group that is reactive with an amine, such as a carboxyl group; on the other hand, the reactive end of the linker to conjugate with the antibody is typically with a basic amine or carboxyl group on the cytotoxin. It is a site that can be conjugated to a cytotoxin through the formation of an amide bond, thus typically a carboxyl or basic amine group. When the term "linker" is used to describe a conjugated form of a linker, one or both of the reactive ends may be between the linker and / or the cytotoxin, and the linker and / or the antibody. Or it does not exist (eg, it has been converted to reactive partial structure Z', chemical partial structure Z) because a bond has been formed between its antigen-binding fragments, or it is in the process of reaction (eg, it is in the process of reaction). Only the carbonyl of the carboxylic acid, etc.). Such conjugating reactions are further described below in the present application.

Various linkers can be used to conjugate the antibodies or antibody fragments described in this application to cytotoxic molecules. In some embodiments, the linker is cleavable under intracellular conditions, so that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet another embodiment, the linker unit is not cleavable and the drug is released, for example, by degradation of the antibody. The linkers useful for the ADCs of the invention are preferably extracellularly stable, prevent ADC molecules from aggregating, and keep the ADC soluble in water-soluble media and in monomeric form. Prior to being transported or delivered into the cell, the ADC is preferably stable and remains intact, i.e., the antibody remains linked to the drug moiety. The linker is stable outside the target cell and may cleave at some effective rate inside the cell. Effective linkers are: (i) maintain the specific binding properties of the antibody; (ii) allow the conjugate or drug moiety to be delivered intracellularly; (iii) the conjugate It remains stable and intact, i.e., not cleaved until delivered or transported to its target site; and (iv) maintain the cytotoxic, cytotoxic or cell proliferation inhibitory effects of the cytotoxic moiety. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC, and LC / MS of separation / analysis techniques. The covalent bond between the antibody and the drug moiety requires the linker to have two reactive functional groups (ie, divalent in the sense of reactivity). Bivalent linker reagents are known to help bind two or more functional or biologically active moieties such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups. A method for obtaining a conjugate is described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p.234-242).

Suitable linkers that can be cleaved include, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage. Examples include those that can be cleaved (see, eg, Leriche et al., Bioorg. Med. Chem., 20: 571-582, 2012, the disclosure of which is a suitable linker for co-conjugated conjugates. As relevant, incorporated by reference in this application). Suitable linkers that can be cleaved include, for example, chemical partial structures such as hydrazine, disulfide, thioether or dipeptide.

Examples of the linker that can be hydrolyzed under acidic conditions include hydrazones, semicarbazones, thiosemicarbazones, cis-aconite amides, ortho esters, acetals, ketals and the like (for example, the United States). Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. The entire linker is incorporated by reference in this application as it relates to a suitable linker to be conjugated to. Such linkers are relatively stable under neutral pH conditions, such as blood conditions, but unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes.

Linkers that can be cleaved under reducing conditions include, for example, disulfides. For example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate) and SMPT. Various disulfide linkers are available, including disulfide linkers that can be formed using (N-succinimidyl-oxycarbonyl-α-methyl-α- (2-pyridyl-dithio) toluene), SPDB and SMPT. Known in the art (eg Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford) See U. Press, 1987. See also US Pat. No. 4,880,935. Each of these disclosures is incorporated herein by reference in its entirety as relating to a linker suitable for covalently conjugating. Is done.

Linkers that are sensitive to enzymatic hydrolysis may be, for example, peptide-containing linkers that are cleaved by intracellular peptidases or protease enzymes, including, but not limited to, lysosomal proteases or endosomal proteases. .. One advantage of utilizing the release of the therapeutic agent by intracellular proteolysis is that the therapeutic agent is usually attenuated when conjugated, and the serum stability of the conjugate is It is usually expensive. In some embodiments, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides or pentapeptides. Examples of suitable peptides include peptides containing amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. Amino acid residues containing the amino acid linker component include naturally occurring ones, as well as minor amino acids and non-naturally occurring amino acid analogs (eg, citrulline). Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valin-citrulin (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker may be Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or. Dipeptides such as Trp-Cit can be mentioned. Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed, for example, in US Pat. No. 6,214,345 (this disclosure relates to linkers suitable for covalently conjugated in its entirety. Is incorporated by reference in this application). In some embodiments, the linker includes a dipeptide selected from Val-Ala and Val-Cit.

Suitable linkers described in this application for conjugating an antibody or antibody fragment to a cytotoxic molecule include linkers capable of releasing cytotoxin by a 1,6-elimination process. The chemical partial structures that enable this desorption process include p-aminobenzyl (PAB) groups, 6-maleimide hexanoic acid, pH-sensitive carbonates, and Jain et al., Pharm. Res. 32: 3256- Other reagents described in 3540, 2015, which are incorporated by reference in their entirety as they relate to linkers suitable for co-binding conjugation, and the like.

In some embodiments, the linker may include a "self-destructing" group such as PAB or PABC (para-aminobenzyloxycarbonyl) described above, which may be described, for example, in Carl et al., J. Med. Chem. (1981) 24: 479-480; Chakravarty et al (1983) J. Med. Chem. 26: 638-644; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; WO98 / 13059 US20040052793; US6677435; US5621002; US20040121940; disclosed in WO2004 / 032828. Other such chemical partial structures (“self-destructing linkers”) capable of this process include methylene carbamate and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines and the like. Linkers containing such heterocyclic self-destructive groups include, for example, US Patent Application Publication 20160303254 and 20150079114, and US Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9: 2237; US 2005. / 0256030; de Groot et al (2001) J. Org. Chem. 66: 8815-8830; and US 7223837. In some embodiments, the dipeptide is used in conjunction with a self-destructing linker.

Suitable linkers for use in this application are C ₁ -C ₆ alkylene, C ₁ -C ₆ heteroalkylene, C ₂ -C ₆ alkenylene, C ₂ -C ₆ hetero alkenylene, C ₂ -C ₆ alkinylene, C ₂ It may further contain one or more groups selected from -C ₆ heteroalkynylene, C ₃ -C ₆ cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which is optional. May have been replaced. Non-restricted examples of such groups include (CH ₂ ) _p , (CH ₂ CH ₂ O) _p , and-(C = O) (CH ₂ ) _p -units, where , P are integers from 1 to 6 and are selected independently for each case.

ここで、aは0又は1である；並びに
R¹⁰は、水素、C₁-C₂₄アルキル基、C₃-C₂₄シクロアルキル基、C₁-C₂₄(ヘテロ)アリール基、C₁-C₂₄アルキル（ヘテロ）アリール基、及びC₁-C₂₄（ヘテロ）アリールアルキル基、C₁-C₂₄アルキル基、C₃-C₂₄シクロアルキル基、C₂-C₂₄（ヘテロ）アリール基、C₃-C₂₄アルキル（ヘテロ）アリール基、及びC₃-C₂₄（ヘテロ）アリールアルキル基、からなる基より選択され、これらの各々は、O、S、及びNR¹¹R¹²より選択される1種以上のヘテロ原子によって、任意選択的に置換している、及び／又は任意選択的に割り込まれていることがある、ここで、R¹¹及びR¹²は、水素及びC₁-C₄アルキル基からなる群から独立して選択される；又はR¹⁰は細胞毒素である、ここで、前記細胞毒素は、スペーサ部分を介して、Nに任意選択的に結合する。そのような基を含むリンカーは、例えば、米国特許9,636,421号及び米国特許出願公開第2017/0298145号(これら開示は、細胞毒素及び抗体又はその抗原結合フラグメントへの共有結合的なコンジュゲーションに適したリンカーに関連するものとして、その全体が本出願に参照により取り込まれる)に記載されている。 Suitable linkers may contain groups that have properties that enhance solubility. For example, a linker containing (CH ₂ CH ₂ O) _p units (polyethylene glycol, PEG) can be highly soluble (eg, substituted with amino, sulfonic acid, phosphonic acid or phosphate residues and alkyled. Can increase the solubility of the chain). Linkers containing such substructures are disclosed, for example, in US Pat. Nos. 8,236,319 and 9,504,756 (each of these disclosures relates to a linker suitable for covalent conjugation in its entirety. Incorporated by reference in this application). Groups that further enhance solubility include, for example, acyl and carbamoylsulfamide groups having the following structures:

Where a is 0 or 1;
R ¹⁰ is hydrogen, C ₁ -C ₂₄ alkyl group, C ₃ -C ₂₄ cycloalkyl group, C ₁ -C ₂₄ (hetero) aryl group, C ₁ -C ₂₄ alkyl (hetero) aryl group, and C _1- C ₂₄ (hetero) arylalkyl groups, C ₁ -C ₂₄ alkyl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₂ -C ₂₄ (hetero) aryl groups, C ₃ -C ₂₄ alkyl (hetero) aryl groups, and Selected from groups consisting of C ₃ -C ₂₄ (hetero) arylalkyl groups, each of which is optionally substituted with one or more heteroatoms selected from O, S, and NR ¹¹ R ¹² . And / or may be optionally interrupted, where R ¹¹ and R ¹² are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl groups; or R ¹⁰ is a cytotoxin, where the cytotoxin optionally binds to N via a spacer moiety. Linkers containing such groups are suitable, for example, in US Pat. No. 9,636,421 and US Patent Application Publication No. 2017/0298145 (these disclosures are suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof. As related to the linker, in its entirety is incorporated by reference in this application).

In some embodiments, the linker is a hydrazine, disulfide, thioether, dipeptide, p-aminobenzyl (PAB) group, heterocyclic self-destructing group, optionally substituted C ₁ -C ₆ alkyl, optional. C ₁ -C ₆ heteroalkyl substituted with, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₂ -C ₆ heteroalkenyl, optionally substituted C ₂ -C ₆ alkynyl, optionally substituted C ₂ -C ₆ heteroalkynyl, optionally substituted C ₃ -C ₆ cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted Includes one or more of substituted aryls, optionally substituted heteroaryls, solubility enhancing groups, acyls,-(C = O)-, or-(CH ₂ CH ₂ O) _p -groups. Sometimes, where p is an integer from 1 to 6. One of skill in the art recognizes that one or more of the listed groups can be present in the form of divalent (diradical) species (eg, C ₁ -C ₆ alkylene, etc.).

である；
L₂は、存在しない、又は-(CH₂)_m-, -NR¹³(CH₂)_m-, -(CH₂)_mNR¹³C(=O)(CH₂)_m-, -X₄, -(CH₂)_mNR¹³C(=O)X₄, - (CH₂)_mNR¹³C(=O)-, -((CH₂)_mO)_n(CH₂)_m-, -((CH₂)_mO)_n(CH₂)_mX₃(CH₂)_m-, - NR¹³((CH₂)_mO)_nX₃(CH₂)_m-, -NR¹³((CH₂)_mO)_n(CH₂)_mX₃(CH₂)_m-, -X₁X₂C(=O)(CH₂)_m-, - (CH₂)_m(O(CH₂)_m)_n-, -(CH₂)_mNR¹³(CH₂)_m-, -(CH₂)_mNR¹³C(=O)(CH₂)_mX₃(CH₂)_m-, - (CH₂)_mC(=O)NR¹³(CH₂)_mNR¹³C(=O)(CH₂)_m-, -(CH₂)_mC(=O)-, - (CH₂)_mNR¹³(CH₂)_mC(=O)X₂X₁C(=O)-, -(CH₂)_mX₃(CH₂)_mC(=O)X₂X₁C(=O)-, - (CH₂)_mC(=O)NR¹³(CH₂)_m-, -(CH₂)_mC(=O)NR¹³(CH₂)_mX₃(CH₂)_m-, -(CH₂)_mX₃(CH₂)_mNR¹³C(=O)(CH₂)_m-, -(CH₂)_mX₃(CH₂)_mC(=O)NR¹³(CH₂)_m-, - (CH₂)_mO)_n(CH₂)_mNR¹³C(=O)(CH₂)_m-, -(CH₂)_mC(=O)NR¹³(CH₂)_m(O(CH₂)_m)_n-, - (CH₂)_m(O(CH₂)_m)_nC(=O)-, -(CH₂)_mNR¹³(CH₂)_mC(=O)-, -(CH₂)_mC(=O)NR¹³(CH₂)_mNR¹³C(=O)-, -(CH₂)_m(O(CH₂)_m)_nX₃(CH₂)_m-, - (CH₂)_mX₃((CH₂)_mO)_n(CH₂)_m-, -(CH₂)_mX₃(CH₂)_mC(=O)-, - (CH₂)_mC(=O)NR¹³(CH₂)_mO)_n(CH₂)_mX₃(CH₂)_m-, - (CH₂)_mX₃(CH₂)_m(O(CH₂)_m)_nNR¹³C(=O)(CH₂)_m-, -(CH₂)_mX₃(CH₂)_m(O(CH₂)_m)_nC(=O)-, -(CH₂)_mX₃(CH₂)_m(O(CH₂)_m)_n-, -(CH₂)_mC(=O)NR¹³(CH₂)_mC(=O)-, -(CH₂)_mC(=O)NR¹³(CH₂)_m(O(CH₂)_m)_nC(=O)-, -((CH₂)_mO)_n(CH₂)_mNR¹³C(=O)(CH₂)_m-, -(CH₂)_mC(=O)NR¹³(CH₂)_mC(=O)NR¹³(CH₂)_m-, -(CH₂)_mNR¹³C(=O)(CH₂)_mNR¹³C(=O)(CH₂) -(CH₂)_mX₃(CH₂)_mC(=O)NR¹³-, -(CH₂)_mC(=O)NR¹³-, -(CH₂)_mX₃-, -C(R¹³)₂(CH₂)_m-, -(CH₂)_mC(R¹³)₂NR¹³-, -(CH₂)_mC(=O)NR¹³(CH₂)_mNR¹³-, - (CH₂)_mC(=O)NR¹³(CH₂)_mNR¹³C(=O)NR¹³-, -(CH₂)_mC(=O)X₂X₁C(=O)-, - C(R¹³)₂(CH₂)_mNR¹³C(=O)(CH₂)_m-, -(CH₂)_mC(=O)NR¹³(CH₂)_mC(R¹³)₂NR¹³-, - C(R¹³)₂(CH₂)_mX₃(CH₂)_m-, -(CH₂)_mX₃(CH₂)_mC(R¹³)₂NR¹³-, -C(R¹³)₂(CH₂)_mOC(=O)NR¹³(CH₂)_m-, -(CH₂)_mNR¹³C(=O)O(CH₂)_mC(R¹³)₂NR¹³-, -(CH₂)_mX₃(CH₂)_mNR¹³-, -(CH₂)_mX₃(CH₂)_m(O(CH₂)_m)_nNR¹³-, -(CH₂)_mNR¹³-, -(CH₂)_mC(=O)NR¹³(CH₂)_m(O(CH₂)_m)_nNR¹³-, -(CH₂)_m(O(CH₂)_m)_nNR¹³-, -(CH₂CH₂O)_n(CH₂)_m-, -(CH₂)_m(OCH₂CH₂)_n;-(CH₂)_mO(CH₂)_m-, -(CH₂)_mS(=O)₂-, - (CH₂)_mC(=O)NR¹³(CH₂)_mS(=O)₂-, -(CH₂)_mX₃(CH₂)_mS(=O)₂-, -(CH₂)_mX₂X₁C(=O)-, -(CH₂)_m(O(CH₂)_m)_nC(=O)X₂X₁C(=O)-, -(CH₂)_m(O(CH₂)_m)_nX₂X₁C(=O)-, - (CH₂)_mX₃(CH₂)_mX₂X₁C(=O)-, -(CH₂)_mX₃(CH₂)_m(O(CH₂)_m)_nX₂X₁C(=O)-, - (CH₂)_mX₃(CH₂)_mC(=O)NR¹³(CH₂)_mNR¹³C(=O)-, -(CH₂)_mX₃(CH₂)_mC(=O)NR¹³(CH₂)_mC(=O)-, -(CH₂)_mX₃(CH₂)_mC(=O)NR¹³(CH₂)_m(O(CH₂)_m)_nC(=O)-, -(CH₂)_mC(=O)X₂X₁C(=O)NR¹³(CH₂)_m-, -(CH₂)_mX₃(O(CH₂)_m)_nC(=O)-, -(CH₂)_mNR¹³C(=O)((CH₂)_mO)_n(CH₂)_m-, -(CH₂)_m(O(CH₂)_m)_nC(=O)NR¹³(CH₂)_m-, -(CH₂)_mNR¹³C(=O)NR¹³(CH₂)_m- 若しくは -(CH₂)_mX₃(CH₂)_mNR¹³C(=O)-である；
ここで、
X₁は、

である；
X₂は、

である；
X₃は、

である；及び
X₄は、

である；
ここで
R¹³は、H及びC₁-C₆アルキルから、それぞれの場合に独立して選択される；
mは、1、2、3、4、5、6、7、8、9及び10から、それぞれの場合に独立して選択される；
nは、1、2、3、4、5、6、7、8、9、10、11、12、13及び14から、それぞれの場合に独立して選択される；
並びに、
ここで、1個のアステリスク(*)は、細胞毒素(例えば、アマトキシン)に結合する位置を示す、及び2個のアステリスク(**)は、反応性置換基Z'又は化学的な部分構造Zに結合する位置を示す（但し、L₁及びL₂の両方ともが存在しない、ということはない、という条件付き）。 In some embodiments, the linker L comprises a portion * -L ₁ L ₂ -**, where:
L ₁ does not exist, or-(CH ₂ ) _m NR ¹³ C (= O)-,-(CH ₂ ) _m NR ¹³ -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m- ,

Is;
L ₂ does not exist, or-(CH ₂ ) _m- , -NR ¹³ (CH ₂ ) _m -,-(CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m- , -X ₄ , -(CH ₂ ) _m NR ¹³ C (= O) X ₄ ,-(CH ₂ ) _m NR ¹³ C (= O)-,-((CH ₂ ) _m O) _n (CH ₂ ) _m -,-( (CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m- , --NR ¹³ ((CH ₂ ) _m O) _n X ₃ (CH ₂ ) _m- , -NR ¹³ ((CH ₂ ) ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m- , -X ₁ X ₂ C (= O) (CH ₂ ) _m -,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n -,-(CH ₂ ) _m NR ¹³ (CH ₂ ) _m -,-(CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m X ₃ (CH ₂ ) _m -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m -,-(CH ₂ ) _m C (= O)-,-(CH ₂ ) _m NR ¹³ (CH) ₂ ) _m C (= O) X ₂ X ₁ C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O) X ₂ X ₁ C (= O)-,-( CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m X ₃ (CH ₂ ) _m -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m -,- (CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m (O (CH ₂ ) ) _m ) _n -,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n C (= O)-,-(CH ₂ ) _m NR ¹³ (CH ₂ ) _m C (= O)-,-( CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) ) _m NR ¹³ C (= O)-,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n X ₃ (CH ₂ ) _m -,-(CH ₂ ) _m X ₃ ((CH ₂ ) _m O ) _n (CH ₂ ) _m -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O)-,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m (O (CH ₂ ) _m ) _n NR ¹³ C (= O) (CH ₂ ) _m- , -(CH ₂ ) _m X ₃ (CH ₂ ) _m (O (CH ₂ ) _m ) _n C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m (O (CH ₂ ) _m ) _n -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m C (= O)-,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m (O (CH) ₂ ) _m ) _n C (= O)-,-((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m -,-(CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m NR ¹³ C (= O) (CH) ₂ )-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O) NR ¹³ -,-(CH ₂ ) _m C (= O) NR ¹³ -,-(CH ₂ ) _m X ₃ -,- C (R ¹³ ) ₂ (CH ₂ ) _m -,-(CH ₂ ) _m C (R ¹³ ) ₂ NR ¹³ -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m NR ^13- ,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m NR ¹³ C (= O) NR ¹³ -,-(CH ₂ ) _m C (= O) X ₂ X ₁ C (= O) -, --C (R ¹³ ) ₂ (CH ₂ ) _m NR ¹³ C (= O) (CH ₂ ) _m -,-(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m C (R ¹³ ) ) ₂ NR ^13- , --C (R ¹³ ) ₂ (CH ₂ ) ) _m X ₃ (CH ₂ ) _m -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (R ¹³ ) ₂ NR ^13- , -C (R ¹³ ) ₂ (CH ₂ ) _m OC (= O) ) NR ¹³ (CH ₂ ) _m -,-(CH ₂ ) _m NR ¹³ C (= O) O (CH ₂ ) _m C (R ¹³ ) ₂ NR ¹³ -,-(CH ₂ ) _m X ₃ (CH ₂ ) ) _m NR ¹³ -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m (O (CH ₂ ) _m ) _n NR ¹³ -,-(CH ₂ ) _m NR ¹³ -,-(CH ₂ ) _m C ( = O) NR ¹³ (CH ₂ ) _m (O (CH ₂ ) _m ) _n NR ¹³ -,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n NR ¹³ -,-(CH ₂ CH ₂ O) _n (CH ₂ ) _m -,-(CH ₂ ) _m (OCH ₂ CH ₂ ) _n ;-(CH ₂ ) _m O (CH ₂ ) _m -,-(CH ₂ ) _m S (= O) _2- , -(CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m S (= O) ₂ -,-(CH ₂ ) _m X ₃ (CH ₂ ) _m S (= O) ₂ -,-(CH) ₂ ) _m X ₂ X ₁ C (= O)-,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n C (= O) X ₂ X ₁ C (= O)-,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n X ₂ X ₁ C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m X ₂ X ₁ C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m (O (CH ₂ ) _m ) _n X ₂ X ₁ C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O) NR ¹³ (CH) ₂ ) _m NR ¹³ C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m C (= O)-,-(CH ₂ ) _m X ₃ (CH ₂ ) _m C (= O) NR ¹³ (CH ₂ ) _m (O (CH ₂ ) _m ) _n C (= O)-,-(CH ₂ ) _m C (= O) X ₂ X ₁ C (= O) NR ¹³ (CH ₂ ) _m -,-( CH ₂ ) _m X ₃ (O (CH ₂ ) _m ) _n C (= O)-,-(CH ₂ ) _m NR ¹³ C (= O) ((CH ₂ ) _m O) _n (CH ₂ ) _m- ,-(CH ₂ ) _m (O (CH ₂ ) _m ) _n C (= O) NR ¹³ (CH ₂ ) _m -,-(CH ₂ ) _m NR ¹³ C (= O) NR ¹³ (CH ₂ ) _m -Or-(CH ₂ ) _m X ₃ (CH ₂ ) _m NR ¹³ C (= O)-;
here,
X ₁ is

Is;
X ₂ is

Is;
X ₃ is

And
X ₄

Is;
here
R ¹³ is independently selected from H and C ₁ -C ₆ alkyl in each case;
m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 in each case;
n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 in each case;
and,
Here, one asterisk (*) indicates a position that binds to a cytotoxin (eg, amatoxin), and two asterisks (**) indicate a reactive substituent Z'or a chemical partial structure Z. Indicates the position to bind to (provided that both L ₁ and L ₂ are not absent).

In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In certain embodiments, the p-aminobenzyl group is located between the cytotoxic drug and the protease cleavage site in the linker. In certain embodiments, the p-aminobenzyl group is part of the p-aminobenzyloxycarbonyl unit. In certain embodiments, the p-aminobenzyl group is part of the p-aminobenzylamide unit.

In some embodiments, the linkers are PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac) -PAB, Phe-Lys-PAB, Phe-Lys (Ac) -PAB, D. -Includes Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

In some embodiments, the linker is a peptide, oligosaccharide,-(CH ₂ ) _p -,-(CH ₂ CH ₂ O) _p- , PAB, Val-Cit-PAB, Val-Ala-PAB, Val. -Lys (Ac) -PAB, Phe-Lys-PAB, Phe-Lys (Ac) -PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB Includes one or more combinations of them.

In some embodiments, the linker comprises a-(C = O) (CH ₂ ) _p -unit, where p is an integer of 1-6.

ここで、波線は、細胞毒素及び反応性部分構造Z'に結合する位置を示す。 In certain embodiments, the linker comprises the following structure:

Here, the wavy line indicates the position of binding to the cytotoxin and the reactive partial structure Z'.

ここで、波線は、細胞毒素及び反応性部分構造Z'に結合する位置を示す。このようなPAB-ジペプチド-プロピオニル・リンカーは、例えば、国際公開第2017/149077号（本出願に参照により取り込まれる）、に開示されている。更に、WO2017/149077に開示されている細胞毒素は、本出願に参照により取り込まれる。 In another specific embodiment, the linker comprises the following structure:

Here, the wavy line indicates the position of binding to the cytotoxin and the reactive partial structure Z'. Such PAB-dipeptide-propionyl linkers are disclosed, for example, in WO 2017/149077 (incorporated by reference in this application). In addition, the cytotoxins disclosed in WO 2017/149077 are incorporated by reference in this application.

In certain embodiments, the linker of the ADC is maleimide caproyl-Val-Ala-para-aminobenzyl (mc-Val-Ala-PAB).

In certain embodiments, the linker of the ADC is maleimide caproyl-Val-Cit-para-aminobenzyl (mc-vc-PAB).

を含む。 In some embodiments, the linker is

including.

In some embodiments, the linker comprises MCC (4- [N-maleimidemethyl] cyclohexane-1-carboxylate).

Linkers useful for combining any one or more of the chemical groups, moieties and features disclosed in this application in a variety of ways to conjugate antibodies and cytotoxins as disclosed in this application. What can be formed will be recognized by some of those skilled in the art. Further linkers useful in conjunction with the compositions and methods described in this application are described, for example, in US Patent Application Publication No. 2015/0218220, the disclosure of which is in its entirety by reference to this application. It is captured.

In certain embodiments, the intermediate, which is the precursor of the linker, is reacted with the drug moiety under suitable conditions. In certain embodiments, the reactive group is used on the drug and / or the intermediate or linker. The product of the reaction between the drug and the intermediate, or derivatized drug, is then reacted with the antibody or antigen binding fragment under suitable conditions. Alternatively, the linker or intermediate may be reacted first with the antibody or derivatized antibody and then with the drug or derivatized drug. Such a conjugation reaction is described here more completely.

Many different reactions can be utilized to covalently bind a linker or drug-linker conjugate to the antibody or antigen-binding fragment thereof. Suitable binding positions on the antibody molecule include amine groups of lysine, free carboxylic acid groups of glutamic acid and aspartic acid, sulfhydryl groups of cysteines, and various moieties of aromatic amino acids. For example, a non-specific covalent bond may be carried out using a carbodiimide reaction to link the carboxy (or amino) group on the compound to the amino (or carboxy) group on the antibody moiety. In addition, bifunctional agents such as dialdehydes or imide esters may be used to attach the amino groups on the compound to the amino groups on the antibody moiety. It is also possible to utilize a Schiff base reaction to bind a drug to a binder. This method involves the periodic acid oxidation of a drug containing glycols or hydroxyl groups, thus forming an aldehyde, which is then reacted with a binder. Binding occurs through the formation of a Schiff base with the amino group of the binder. Isothiocyanate may also be used as a coupling agent to covalently bind the drug to the binder. Other techniques are known to those of skill in the art and are within the scope of the present invention.

As described in this application, the linker useful for conjugation to an antibody or antigen binding fragment is, but is not limited to, a chemical formed by a coupling reaction as shown in Table 1 below. Examples include linkers containing partial structure Z. The curves indicate the positions that bind to the antibody or antigen binding fragment and the cytotoxic molecule, respectively.

Some of those skilled in the art have a covalent coupling reaction in which the reactive substituent Z'bound to the linker and the reactive substituent on the antibody or antigen-binding fragment thereof form a chemical partial structure Z. Recognize that it is involved in the above-mentioned reactive partial structure Z'. Thus, antibody-drug conjugates useful in conjunction with the methods described in this application may be formed by the reaction of an antibody, or antigen-binding fragment thereof, with a linker or cytotoxin-linker conjugate. As described in this application, the linker or cytotoxin-linker conjugate comprises a reactive substituent Z'and the like and is suitable for reaction with an antibody or a reactive substituent on an antigen-binding fragment thereof. , Form a chemical partial structure Z.

である；
ここで、
R¹³は、H及びC₁-C₆アルキルから、それぞれの場合に独立して選択される；
R¹⁴は、-S(CH₂)_nCHR¹⁵NHC(=O)R¹³である；
R¹⁵は、R¹³又は-C(=O)OR¹³である；
R¹⁶は、H、C₁-C₆アルキル、F、Cl、及びOHから、それぞれの場合に独立して選択される；
R¹⁷は、H、C₁-C₆アルキル、F、Cl 、-NH₂、-OCH₃、-OCH₂CH₃、-N(CH₃)₂、-CN、-NO₂ 及び-OHから、それぞれの場合に独立して選択される；並びに、
R¹⁸は、H、C₁-C₆アルキル、F、-C(=O)OHで置換されたベンジルオキシ、-C(=O)OHで置換されたベンジル、-C(=O)OHで置換されたC₁-C₄アルコキシ基、及び-C(=O)OHで置換されたC₁-C₄アルキル基から、それぞれの場合に独立して選択される。 In some embodiments, Z'is -NR ¹³ C (= O) CH = CH ₂ , -N ₃ , -SH, -S (= O) ₂ (CH = CH ₂ ),-(CH ₂ ). ₂ S (= O) ₂ (CH = CH ₂ ), -NR ¹³ S (= O) ₂ (CH = CH ₂ ), -NR ¹³ C (= O) CH ₂ R ¹⁴ , -NR ¹³ C (= O) ) CH ₂ Br, -NR ¹³ C (= O) CH ₂ I, -NHC (= O) CH ₂ Br, -NHC (= O) CH ₂ I, -ONH ₂ , -C (O) NHNH ₂ ,- CO ₂ H, -NH ₂ , -NH (C = O), -NC (= S),

Is;
here,
R ¹³ is independently selected from H and C ₁ -C ₆ alkyl in each case;
R ¹⁴ is -S (CH ₂ ) _n CHR ¹⁵ NHC (= O) R ¹³ ;
R ¹⁵ is R ¹³ or -C (= O) OR ¹³ ;
R ¹⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, and OH in each case;
R ¹⁷ is derived from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N (CH ₃ ) ₂ , -CN, -NO ₂ and -OH. Selected independently in each case;
R ¹⁸ is H, C ₁ -C ₆ alkyl, F, -C (= O) OH substituted benzyloxy, -C (= O) OH substituted benzyl, -C (= O) OH. It is independently selected from the substituted C ₁ -C ₄ alkoxy group and the C ₁ -C ₄ alkyl group substituted with -C (= O) OH in each case.

As shown in Table 31, examples of suitable reactive substituents on said linkers and antibodies, or antigen-binding fragments thereof, include nucleophilic / nucleophilic pairs (eg, thiol / haloalkyl pairs, amine / carbonyl pairs, etc. Or thiol / α, β-unsaturated carbonyl pair), diene / dienophile pair (eg, in particular, azide / alkyne pair, or diene / α, β-unsaturated carbonyl pair) and the like. Coupling reactions between reactive substituents to form the chemical partial structure Z are, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine. Condensation, hydrazine formation, amidation, esterification, disulfide formation, cyclization addition (eg, among others, [4 + 2] Diels-Alder cyclization addition, [3 + 2] Huisgen cyclization addition), aromatic nucleophilic substitution , Aromatic electrophilic substitutions, and other reaction modalities known in the art or described in this application. Preferably, the linker comprises an electrophilic functional group for reaction with the nucleophilic functional group on the antibody or antigen-binding fragment thereof.

As disclosed in this application, the reactive substituents that may be present in the antibody or its antigen-binding fragment are, but are not limited to, (i) N-terminal amine groups, (ii) side chain amines. A nucleophilic group such as a group (eg, lysine), (iii) side chain thiol group (eg, cysteine), and (iv) sugar hydroxyl group or amino group (where the antibody is glycosylated) Can be mentioned. As disclosed in this application, reactive substituents that may be present in an antibody or antigen-binding fragment thereof are, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; lysine residues. Amino moieties; carboxyl moieties of aspartic acid and glutamate residues; and thiol moieties of cysteine residues, as well as the non-natural amino acids propargyl, azide, haloaryl (eg, fluoroaryl), haloheteroaryl (eg, fluoroheteroaryl). ), Haloalkyl, and haloheteroalkyl moieties. In some embodiments, as disclosed in this application, reactive substituents present within an antibody, or antigen-binding fragment thereof, include amine or thiol moieties. Certain antibodies have reducible interchain disulfides (ie, cysteine crosslinks). The antibody can be made reactive in order to be conjugated to a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Therefore, each cysteine crosslink theoretically forms two reactive thiol nucleophiles. The reaction of lysine with 2-iminothiorane (a trout reagent) can introduce additional nucleophiles into the antibody to convert the amine to a thiol. By introducing one, two, three, four, or more cysteine residues (eg, by preparing a mutant antibody containing one or more originally absent cysteine amino acid residues). , Reactive thiol groups may be introduced into the antibody (or fragment thereof). US Pat. No. 7,521,541 teaches engineered antibodies by introducing reactive cysteine amino acids.

In some embodiments, the reactive partial structure Z'bound to the linker is a nucleophilic group that is reactive with the electrophilic group present on the antibody. Useful electrophilic groups on antibodies include, but are not limited to, carbonyl groups of aldehydes and ketones. The heteroatom of the nucleophilic group may react with the nucleophilic group on the antibody to form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

In some embodiments, Z is a reaction between a reactive nucleophilic substituent (eg, an amine and thiol moiety) present within the antibody or its antigen-binding fragment, and a reactive electrophilic substituent Z'. Is a product of. For example, Z'can be, in particular, a Michael acceptor (eg, maleimide), an activated ester, an electron-deficient carbonyl compound, and an aldehyde.

For example, suitable linkers for the synthesis of ADCs include, but are not limited to, reactive substituent Z'such as a maleimide or haloalkyl group. These include reagents (eg, especially succinimidyl 4- (N-maleimidemethyl) -cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidebenzoyl-N- Hydroxysuccinimidyl esters (MBS), sulfo-MBS, and succinimidyl iodoacetates, etc., eg, described in Liu et al., 18: 690-697, 1979 [this disclosure is a chemistry. Incorporated by reference in this application as relating to a linker for conjugation]), it may bind to said linker.

In some embodiments, the reactive substituent Z'attached to linker L is maleimide, azide, or alkyne. An example of a maleimide-containing linker is a non-cleavic maleimide caproyl-based linker, which is particularly useful for conjugating microtubule disruptors such as auristatins. Such linkers are described in Doronina et al., Bioconjugate Chem. 17: 14-24, 2006 (this disclosure is incorporated by reference in this application as relating to linkers for chemical conjugates. ).

In some embodiments, the reactive substituent Z'is-(C = O)-or -NH (C = O)-, so that the linkers are-(C = O)-or, respectively. The partial structure of the amide or urea resulting from the reaction of the -NH (C = O) -group with the amino group of the antibody or its antigen-binding fragment may bind to the antibody or its antigen-binding fragment.

In some embodiments, the reactive substituent is an N-maleimidyl group, a halogenated N-alkylamide group, a sulfonyloxyN-alkylamide group, a carbonate group, a halide sulfonyl group, a thiol group or a derivative thereof, internally. Alkinyl group containing a carbon-carbon triple bond, (hetero) cycloalkynyl group, bicyclo [6.1.0] non-4-in-9-yl group (bicyclo [6.1.0] non-4-yn-9- yl group), alkenyl group containing carbon-carbon double bond inside, cycloalkenyl group, tetrazinyl group, azido group, phosphine group, nitrile oxide group, nitron group, nitrile imine group, diazo group, ketone group, (O-alkyl) hydroxylamino group, hydrazine group, halogenated N-maleimidyl group, 1,1-bis (sulfonylmethyl) methylcarbonyl group or desorbed derivative thereof, halogenated carbonyl group, or allenamide group, which are these. Each may be replaced arbitrarily. In some embodiments, the reactive substituent comprises a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero) cycloalkynyl group.

Non-limited examples of amatoxin-linker conjugates comprising a reactive substituent Z'suitable for reaction with a reactive residue on the antibody or its antigen-binding fragment are not limited. 7'C-(4- (6- (maleimide) hexanoyl) piperazin-1-yl) -amatoxin; 7'C-(4- (6- (maleimide) hexaneamide) piperidine-1- Il) -amatoxin; 7'C- (4- (6- (6- (maleimide) hexaneamide) hexanoyl) piperazine-1-yl) -amatoxin; 7'C- (4- (4- ((maleimide) methyl) ) Cyclohexanecarbonyl) piperazin-1-yl) -amatoxin; 7'C-(4-(6-(4-((maleimide) methyl) cyclohexanecarboxamide) hexanoyl) piperazin-1-yl) -amatoxin; 7'C- (4- (2- (6- (maleimide) hexaneamide) ethyl) piperazine-1-yl) -amatoxin; 7'C- (4- (2- (6- (6- (maleimide) hexaneamide)) hexaneamide ) Ethyl) piperazine-1-yl) -amatoxin; 7'C-(4-(2-(4-((maleimide) methyl) cyclohexanecarboxamide) ethyl) piperazine-1-yl) -amatoxin; 7'C-( 4- (2- (6- (4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) ethyl) piperazine-1-yl) -amatoxin; 7'C- (4- (2- (3-carboxypropaneamide) ) Ethyl) piperazine-1-yl) -amatoxin; 7'C-(4- (2- (2-bromoacetamide) ethyl) piperazine-1-yl) -amatoxin; 3- (Pyridin-2-yldisulfanyl) propanamide) ethyl) piperazine-1-yl) -amatoxin; 7'C- (4- (2- (4- (maleimide) butaneamide) ethyl) piperazine-1-yl) -Amatoxin; 7'C- (4- (2- (maleimide) acetyl) piperazin-1-yl) -amatoxin; 7'C-(4- (3- (maleimide) propanoyl) piperazin-1-yl)-amatoxin 7'C-(4- (4- (maleimide) butanoyl) piperazin-1-yl) -amatoxin; 7'C- (4- (2- (6- (4-((maleimide) methyl) cyclohexanecarboxamide)) Hexaamide) ethyl) piperi Jin-1-yl) -amatoxin; 7'C-(3-((6- (maleimide) hexaneamide) methyl) pyrrolidine-1-yl) -amatoxin; 7'C- (3-((6- (6) 6) -(Maleimide) hexaneamide) hexaneamide) methyl) pyrrolidine-1-yl) -amatoxin; 7'C-(3-((4-((maleimide) methyl) cyclohexanecarboxamide) methyl) pyrrolidine-1-yl)- Amatoxin; 7'C-(3-((6-((4- (maleimide) methyl) cyclohexanecarboxamide) hexaneamide) methyl) pyrrolidine-1-yl) -amatoxin; 7'C- (4- (2- (2- ( 6- (2- (Aminooxy) acetamide) hexaneamide) ethyl) piperidin-1-yl) -amatoxin; 7'C- (4- (2- (4- (2- (aminooxy) acetamide) butaneamide) ethyl ) Piperidine-1-yl) -amatoxin; 7'C- (4- (4- (2- (aminooxy) acetamide) butanoyl) piperazin-1-yl) -amatoxin; 7'C- (4- (6- (6- (6- (6-) 6-) (2- (Aminooxy) acetamide) hexanoyl) piperazin-1-yl) -amatoxin; 7'C-((4- (6- (maleimide) hexaneamide) piperidin-1-yl) methyl) -amatoxin; 7' C-((4- (2- (6- (maleimide) hexaneamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4- (6- (maleimide) hexanoyl) piperazin-1 -Il) Methyl) -amatoxin; (R) -7'C-((3-((6- (maleimide) hexaneamide) methyl) pyrrolidine-1-yl) methyl)-amatoxin; (S) -7'C -((3-((6- (maleimide) hexaneamide) methyl) pyrrolidine-1-yl) methyl) -amatoxin; 7'C-((4- (2- (6- (6- (maleimide) hexaneamide) hexaneamide ) Hexaamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (4-((maleimide) methyl) cyclohexanecarboxamide) ethyl) piperidin-1-yl) methyl) -Amatoxin; 7'C-((4- (2- (6- (4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) ethyl) piperidin-1-yl) methyl)-amatoxin; 7'C-( (4- (2- (6- (maleimide) hexaneamide) Ethyl) piperazin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (6- (6- (maleimide) hexaneamide) hexaneamide) ethyl) piperazin-1-yl) methyl)- Amatoxin; 7'C-((4- (2- (4-((maleimide) methyl) cyclohexanecarboxamide) ethyl) piperazine-1-yl) methyl)-amatoxin; 7'C-((4- (2-() 6-(4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) ethyl) piperazin-1-yl) methyl) -amatoxin; 7'C-((3-((6- (6- (maleimide) hexaneamide) ) Hexaamide) -S-methyl) pyrrolidine-1-yl) methyl) -amatoxin; 7'C-((3-((6- (6- (maleimide) hexaneamide) hexaneamide) -R-methyl) pyrrolidine) -1-yl) Methyl) -amatoxin; 7'C-((3-((4-((maleimide) methyl) cyclohexanecarboxamide) -S-methyl) pyrrolidine-1-yl) methyl) -amatoxin; 7'C -((3-((4-((maleimide) methyl) cyclohexanecarboxamide) -R-methyl) pyrrolidine-1-yl) methyl)-amatoxin; 7'C-((3-((6- (4- (4-( (Maleimide) Methyl) Cyclohexanecarboxamide) Hexamide) Methyl) Pyrrolidine-1-yl) Methyl) -amatoxin; 7'C-((4- (2- (3-carboxypropanamide) ethyl) piperazin-1-yl) Methyl) -amatoxin; 7'C-((4- (6- (6- (maleimide) hexaneamide) hexanoyl) piperazine-1-yl) methyl) -amatoxin; 7'C-((4- (6- (6-(6-( 4-((maleimide) methyl) cyclohexanecarboxamide) hexanoyl) piperazin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (maleimide) acetyl) piperazin-1-yl) methyl) -amatoxin 7'C-((4- (3- (maleimide) propanoyl) piperazin-1-yl) methyl) -amatoxin; 7'C-((4- (4- (maleimide) butanoyl) piperazin-1-yl)) Methyl) -amatoxin; 7'C-((4- (2- (2- (maleimide) acetamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (4)) -(Maleimide) Butanamide) Ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (6- (4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) ethyl) piperidin-1-yl) Methyl) -amatoxin; 7'C-((3-((6- (maleimide) hexaneamide) methyl) azetidine-1-yl) methyl) -amatoxin; 7'C-((3- (2- (6- (6- (6-) 6-) (Maleimide) Hexamide) Ethyl) Azetidine-1-yl) Methyl) -amatoxin; 7'C-((3-((4-((maleimide) Methyl) Cyclohexanecarboxamide) Methyl) Azetidine-1-yl) Methyl) -Amatoxin; 7'C-((3- (2- (4-((maleimide) methyl) cyclohexanecarboxamide) ethyl) azetidine-1yl) methyl)-amatoxin; 7'C-((3- (2-(- 6-(4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) ethyl) azetidine-1-yl) methyl) -amatoxin; 7'C-(((2- (6- (maleimide) -N-methylhexane) Amide) ethyl) (methyl) amino) methyl) -amatoxin; 7'C-(((4- (6- (maleimide) -N-methylhexaneamide) butyl (methyl) amino) methyl) -amatoxin; 7'C -((2- (2- (6- (maleimide) hexaneamide) ethyl) aziridine-1-yl) methyl) -amatoxin; 7'C-((2- (2- (6- (4-((maleimide) maleimide) ) Methyl) cyclohexanecarboxamide) hexaneamide) ethyl) aziridine-1-yl) methyl) -amatoxin; 7'C-((4- (6- (6- (2- (aminooxy) acetamide) hexaneamide) hexanoyl) Piperazin-1-yl) Methyl) -amatoxin; 7'C-((4- (1- (aminooxy) -2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oil) piperazine) -1-yl) Methyl) -amatoxin; 7'C-((4- (2- (2- (aminooxy) acetamide) acetyl) piperazine-1-yl) methyl) -amatoxin; 7'C-((4) -(3- (2- (Aminooxy) acetamide) propanoyl) piperazin-1-yl) methyl) -amatoxin; 7'C-((4- (4- (2- (aminooxy) acetamide) butanoyl) piperazin- 1-Il) Methyl) -A Matoxin; 7'C-((4- (2- (6- (2- (aminooxy) acetamide) hexaneamide) ethyl) piperidin-1-yl) methyl)-amatoxin; 7'C-((4- ((4- ( 2- (2- (2- (Aminooxy) acetamide) acetamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (4- (2- (aminooxy)) Acetamide) butaneamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4- (20- (aminooxy) -4,19-dioxo-6,9,12,15-tetraoxa-3) , 18-diazycocil) piperidin-1-yl) methyl) -amatoxin; 7'C-(((2- (6- (2- (aminooxy) acetamide) -N-methylhexaneamide) ethyl) (methyl) amino ) Methyl) -amatoxin; 7'C-(((4- (6- (2- (aminooxy) acetamide) -N-methylhexaneamide) butyl) (methyl) amino) methyl)-amatoxin; 7'C- ((3-((6- (4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) methyl) pyrrolidine-1-yl) -S-methyl) -amatoxin; 7'C-((3-((6) -(4-((maleimide) methyl) cyclohexanecarboxamide) hexaneamide) -R-methyl) pyrrolidine-1-yl) methyl) -amatoxin; 7'C-((4- (2- (2-bromoacetamide) ethyl) ) Piperazin-1-yl) methyl) -amatoxin; 7'C-((4- (2- (2-bromoacetamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 7'C-((4-4-yl) methyl) (2- (3- (Pridin-2-yldisulfanyl) propanamide) ethyl) piperidin-1-yl) methyl) -amatoxin; 6'O-(6- (6- (maleimide) hexaneamide) hexyl) -amatoxin 6'O-(5-(4-((maleimide) methyl) cyclohexanecarboxamide) pentyl) -amatoxin; 6'O-(2-((6- (maleimide) hexyl) oxy) -2-oxoethyl) -amatoxin 6'O-((6- (maleimide) hexyl) carbamoyl) -amatoxin; 6'O-((6- (4-((maleimide) methyl) cyclohexanecarboxamide) hexyl) carbamoyl)-amatoxin; 6'O- (6- (2-bromoacetamide Do) hexyl) -amatoxin; 7'C- (4- (6- (azido) hexaneamide) piperidin-1-yl) -amatoxin; 7'C- (4- (hex-5-inoylamino) piperidin-1 -Il) -amatoxin; 7'C- (4- (2- (6- (maleimide) hexaneamide) ethyl) piperazin-1-yl) -amatoxin; 7'C- (4- (2- (6- (6- (6-( 6- (maleimide) hexaneamide) hexaneamide) ethyl) piperazin-1-yl) -amatoxin; 6'O-(6- (6- (11,12-didehydro-5,6-dihydro-dibenz) [b, f ] Azosin-5-yl) -6-oxohexaneamide) hexyl) -amatoxin; 6'O-(6- (hex-5-inoylamino) hexyl) -amatoxin; Aminooxy) acetylamide) hexyl) -amatoxin; 6'O-((6-aminooxy) hexyl) -amatoxin; and 6'O-(6- (2-iodoacetamide) hexyl) -amatoxin.

ここで、Sは、CD45に結合する、抗体、又はその抗原結合フラグメント内に存在する反応性置換基を表す硫黄原子である(例えば、システイン残基の-SH基に由来する)。 In some embodiments, the chemical partial structure Z is selected from Table 1. In some embodiments, the chemical partial structure Z is

Here, S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD45 (eg, derived from the -SH group of a cysteine residue).

。 In some embodiments, the structure L-Z'of the linker-reactive substituent prior to conjugation with the antibody or antigen-binding fragment thereof is:

..

。 In some embodiments, the amatoxin disclosed in this application is conjugated to a linker-reactive substructure-L-Z'having the following formula:

..

。 In some embodiments, the amatoxin disclosed in this application is conjugated to a linker-reactive substructure-L-Z'having the following formula:

..

Particularly useful in combination with the compositions and methods described in this application are the above linker substructures and amatoxin-linker conjugates, eg, US Patent Application Publication No. 2015/0218220 and Patent Application Publication No. WO 2017. 1 / 149077, the entire disclosure of each of these is incorporated herein by reference in its entirety.

Preparation of antibody-drug conjugate In the ADC of formula I disclosed in this application, the anti-CD45 antibody, or antigen-binding fragment thereof, is one via the linker L and the chemical partial structure Z disclosed in this application. Conjugate to the above cytotoxic drug moiety (D) (eg, about 1 to about 20 drug moieties per antibody). The ADCs of the present disclosure can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those of skill in the art, including: (1) Reactive substituents on antibodies or antigen-binding fragments thereof. Is reacted with a divalent linker reagent to form Ab-ZL as described above in the present application, followed by reaction with drug moiety D; or (2) reactive substituents on the drug moiety, 2 It is reacted with a valent linker reagent to form DL-Z', followed by reaction with a reactive substituent of an antibody or antigen-binding fragment thereof as described above in the present application. Further methods for preparing the ADC are described in this application.

In another embodiment, the anti-CD45 antibody, or antigen-binding fragment thereof, has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The ADC is then formed by conjugating via a sulfur atom of a sulfhydryl group as described above in the present application. Reagents that can be used to modify lysine include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolan hydrochloride (Traut's reagent).

In another embodiment, the anti-CD45 antibody, or antigen-binding fragment thereof, may have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. .. The ADC is then formed by conjugating via a sulfur atom of a sulfhydryl group as described above in the present application.

In yet another embodiment, the anti-CD45 antibody may have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (eg, Laguzza, et al. , J. Med. Chem. 1989, 32 (3), 548-55). The ADC is then formed by conjugating with the corresponding aldehyde as described above in the present application. Other protocols for modifying proteins to bind or associate cytotoxins are described in Colligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002) (in this application). Incorporated by reference).

Methods for conjugating a linker-drug moiety to a cell-targeting protein such as an antibody, immunoglobulin or fragment thereof may be described, for example, in US Pat. No. 5,208,020; US Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006. 034488 (all of which are expressly incorporated by reference in this application in their entirety).

Alternatively, a fusion protein containing the antibody and a cytotoxic drug may be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may include each region encoding two parts of the conjugate adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate. be.

The ADCs described in this application may be administered to a patient (eg, a human patient suffering from an autoimmune disease or cancer) in various dosage forms. For example, the ADC described in this application may be administered to a patient suffering from an autoimmune disease or cancer in the form of an aqueous solution (eg, an aqueous solution containing one or more pharmaceutically acceptable excipients). I have something to do. Suitable pharmaceutically acceptable excipients for use with the compositions and methods described in this application include viscosity modifiers. The aqueous solution may be sterilized using techniques known in the art.

Pharmaceutical formulations containing the anti-CD45 ADCs as described in this application are made by mixing such ADCs with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition). , Osol, A. Ed. (1980)), prepared in the form of lyophilized or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dose and concentration used, including, but not limited to, phosphates, citrus. Buffers such as acid salts and other organic acids; antioxidants containing ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalconium chloride; benzethonium chloride; phenol, butyl or benzyl Alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptide; serum albumin, gelatin, Or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars such as glucose, mannose, or dextrin. Chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or polyethylene glycol (PEG) and the like. Non-ionic surfactant.

Method of treatment
CD45 is an important cell surface molecule that is widely expressed throughout the hematopoiesis and immune system. Described in this application are anti-CD45 antibodies and anti-CD45 antibodies that can be used to treat patients with leukemia and lymphoma, as well as patients with autoimmune diseases such as multiple sclerosis and sclerosis. It is a CD45 ADC. In addition, currently for compositions and methods for promoting engraftment of extrinsic hematopoietic stem cell transplants so that the multi-potency and hematopoietic functionality of these cells are preserved after transplantation. There is a need. The compositions disclosed in this application further provide a solution to this difficult problem.

Therefore, methods for treating various disorders (eg, cell-type disorders in the hematopoietic lineage, cancer, autoimmune disorders, metabolic disorders, stem cell disorders, etc.) are further disclosed in this application. By the compositions and methods described in this application, a population of cells that cause pathology, such as (i) a population of cancer cells (eg, leukemia cells) and autoimmune cells (eg, self-reactive T-cells), is directly expressed. Of endogenous hematopoietic stem cells so that engraftment of the transplanted hematopoietic stem cells is promoted by providing a niche that can be reduced and / or (ii) the transplanted cells can be homing. The population can be reduced. The above activity is obtained by administration of an ADC, antibody, or antigen-binding fragment thereof capable of binding to an antigen expressed by the disease-causing endogenous cell or hematopoietic stem cell. When treating the disease directly, this administration can reduce the amount of cells that give rise to the condition of interest. In cases preparing patients for hematopoietic stem cell transplantation therapy, this administration selectively reduces the population of endogenous hematopoietic stem cells, thereby opening up space in hematopoietic tissues such as bone marrow. Created and its vacancy may then be filled with transplanted extrinsic hematopoietic stem cells. The present invention is based in part on the finding that an ADC, antibody, or antigen-binding fragment thereof capable of binding to CD45 expressed by hematopoietic stem cells can be administered to a patient to obtain the effects of both of the above activities. There is. An ADC, antibody, or antigen-binding fragment that binds to an antigen expressed by hematopoietic stem cells (eg, CD45) to directly reduce the population of cancerous or autoimmune cells, cancer, or autoimmune disease. It may be administered to patients suffering from hematopoietic stem cell transplantation therapy, and may be administered to patients who require hematopoietic stem cell transplantation therapy in order to promote the survival and engraftment ability of transplanted hematopoietic stem cells.

As described in this application, hematopoietic stem cell transplantation therapy may be prescribed to a subject in need of treatment in order to engraft or regenerate one or more blood cell types. Hematopoietic stem cells generally exhibit multi-potency, and thus, but are not limited to, granulocytes (eg, promyelinocytes, neutrophils, eosinophils, eosinophils), erythrocytes (e.g.). For example, reticular erythrocytes, erythrocytes), platelets (thrombocytes) (eg, megakaryocytes, platelet-producing megakaryocytes, platelets), monospheres (eg, monospheres, macrophages), dendritic cells, microglia, It can differentiate into a variety of different blood lines, including bone-breaking cells and lymphocytes (eg, NK cells, B-cells and T-cells) and the like. Hematopoietic stem cells are also capable of self-renewal and thus can give rise to daughter cells with the same potential as mother cells, and are also reintroduced into transplant recipients, homing into the hematopoietic stem cell niche and producing. It is characterized by the ability to reestablish targeted and sustained hematopoiesis.

Therefore, hematopoietic stem cells may be administered to patients who are defective or deficient in one or more cell types of the hematopoietic lineage in vivo to reconstitute the defective or deficient cell population. Allows treatment of pathological conditions associated with defects or deficiencies in an endogenous blood cell population. Therefore, the compositions and methods described in this application are selected from the group consisting of non-malignant hemoglobinopathy (eg, sickle cell anemia, thalassemia, fanconi anemia, aplastic anemia, and Wiscot-Aldrich syndrome). May be used to treat (abnormal hemoglobinopathy). Further, or instead, the compositions and methods described in this application may be used to treat immunodeficiencies such as congenital immunodeficiency. Further, or instead, the compositions and methods described in this application may be used to treat acquired immunodeficiency (eg, acquired immunodeficiency selected from the group consisting of HIV and AIDS). be. The compositions and methods described in this application are metabolic disorders selected from the group consisting of metabolic disorders (eg, glycogen accumulation, mucopolysaccharidosis, Gaucher's disease, Harler's disease, sphingolipidosis, and metachromatic leukodystrophy). ) May be used to treat.

Further, or instead, the compositions and methods described in this application can be used to treat malignant tumors or proliferative disorders such as hematological malignancies, myeloproliferative disorders. In the case of cancer treatment, the compositions and methods described in this application may be administered to the patient prior to hematopoietic stem cell transplantation therapy to reduce the population of endogenous hematopoietic stem cells, in which case the transplanted cells. Can home to the niches created by the steps of reducing endogenous cells and establish productive hematopoiesis. This in turn allows the reconstitution of cell populations that have diminished in the process of eradicating cancer cells, such as in the process of systemic chemotherapy. Exemplary blood cancers that can be treated using the compositions and methods described in this application are, but are not limited to, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic. Includes lymphocytic leukemia, multiple myelogenous tumors, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma, as well as other cancer symptoms including neuroblastoma.

Further diseases that can be treated with the compositions and methods described in this application are, but are not limited to, adenosine deaminase deficiency and severe complex immunodeficiency, hyperimmunoglobulin M syndrome, Chediac-. East disease, hereditary lymphohistiocytosis, marble bone disease, osteodysplasia, storage disease, salacemia major, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis Can be mentioned.

Antibodies described in this application, or antigen-binding fragments thereof, and conjugates may be used to induce transplant tolerance in solid organs. For example, the compositions and methods described in this application may be used to reduce or deplete a population of cells from a target tissue (eg, reduce hematopoietic stem cells from a bone marrow stem cell niche). After such depletion of cells from the target tissue, a population of stem cells or progenitor cells from the organ donor (eg, hematopoietic stem cells from the organ donor) may be administered to the transplant recipient. And after engraftment of such stem cells or progenitor cells, it becomes a temporary or stable mixed chimera, which enables long-term transplanted organ tolerance without the need for further immunosuppressive agents. For example, the compositions and methods described in this application may be used to induce transplant tolerance in solid organ transplant recipients (eg, kidney transplants, lung transplants, liver transplants, and heart transplants, among others). The compositions and methods described in this application induce transplantation tolerance of solid organs, for example, because long-term tolerance of transplanted organs is sufficiently induced even with a low percentage of temporary or stable donor engraftment. Well suited for the purpose of use in particular.

In addition, the compositions and methods described in this application may be used to directly treat cancers such as cancers characterized by cells that are CD45 +. For example, the compositions and methods described in this application may be used to treat leukemia (eg, leukemia in patients with CD45 + leukemia cells). By reducing CD45 + cancerous cells (eg, leukemic cells), the compositions and methods described in this application can be used to directly treat a variety of cancers. Illustrative cancers that may be treated in this manner include acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, And blood cancers such as non-Hodgkin's lymphoma.

In addition, the compositions and methods described in this application may be used to treat autoimmune disorders. For example, an antibody, or antigen-binding fragment thereof, may be administered to a subject such as a human patient suffering from an autoimmune disorder in order to damage CD45 + immune cells. For example, a CD45 + immune cell may be a T-cell-like autoreactive lymphocyte that expresses a T-cell receptor that specifically binds to the self-antigen and initiates an immune response to the self-antigen. be. By reducing autoreactive CD45 + cells, the compositions and methods described in this application may be used to treat autoimmune conditions as described below. In addition, or instead, the compositions and methods described in this application may be used to treat autoimmune diseases by reducing the population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation therapy. In that case, the transplanted cells can be homing to the niche created by the step of reducing endogenous cells and establishing productive hematopoiesis. This in turn allows the population of cells to be reduced during the eradication of autoimmune cells to be reconstituted.

Autoimmune diseases that can be treated using the compositions and methods described in this application are, but are not limited to, psoriasis, psoriatic arthritis, type 1 diabetes (type 1 diabetes), rheumatoid arthritis (RA). ), Human systemic erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute diffuse encephalomyelitis (ADEM), Azison's disease, generalized alopecia , Tonic spondylitis, antiphospholipid antibody syndrome (APS), poor regeneration anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune internal ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS) , Autoimmune ovarian inflammation, Barrow's disease, Bechet's disease, Bullous cyst, Myocardium, Shark's disease, Chronic fatigue immunodeficiency syndrome (CFIDS), Chronic inflammatory demyelinating polyneuropathy, Crohn's disease, Scarring heaven Blisters, Celiac sprue vesicular dermatitis, cold agglutinosis, CREST syndrome, Degos's disease, discoid erythematosus, autoimmune disorders, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fiber Myitis, Good Pasture Syndrome, Graves' Disease, Gillan-Valley Syndrome (GBS), Hashimoto Thyroiditis, Purulent Sweat Adenitis, Idiopathic and / or Acute Thrombocytopenic Purpura, Idiopathic Pulmonary Fibrosis, IgA Neuropathy, Interstitial Bladder inflammation, juvenile arthritis, Kawasaki disease, squamous lichen, Lime's disease, Meniere's disease, mixed connective tissue disease (MCTD), severe myasthenia, neuromyotonia, opsocronus-myocronus syndrome (OMS), optic neuritis, ordo Ord's thyroiditis, scoliosis vulgaris, malignant anemia, polychondritis, polymyositis and dermatomyitis, primary biliary cirrhosis, nodular polyarteritis, polyendocrine gland syndrome, rheumatic polymyopathy, Primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Schegren's syndrome, Stiff-Person's syndrome, hyperan arteritis, temporal arteritis (as giant cell arteritis) Also known), ulcerative colitis, collagenous fibrous colitis, vegetative membrane inflammation, vasculitis, leukoplakia, genital pain (extrinsic vestibular inflammation), and Wegener's granulomatosis.

In some embodiments, the graft is allogeneic. In some embodiments, the implant is autologous.

In some embodiments, the transplant is a bone marrow transplant, a peripheral blood transplant, a cord blood transplant.

In some embodiments, the implant comprises hematopoietic cells (eg, hematopoietic stem cells).

In any of the embodiments described in this application, the graft may be any solid organ or skin graft. In some embodiments, the transplant is selected from the group consisting of kidney transplants, heart transplants, liver transplants, pancreatic transplants, lung transplants, small bowel transplants, and skin transplants.

Dosage Routes and Methods of Dosing The antibodies described in this application, their antigen-binding fragments, or ADCs in various dosage forms suffer from patients (eg, cancer, autoimmune disease, or require hematopoietic stem cell transplantation therapy). May be administered to human patients). For example, an antibody, an antigen-binding fragment thereof, or an ADC described in this application may be used in an aqueous solution (eg, one or more) in a patient suffering from cancer, an autoimmune disease, or in need of hematopoietic stem cell transplantation therapy. It may be administered in the form of an aqueous solution containing a pharmaceutically acceptable excipient). Pharmaceutically acceptable excipients for use with the compositions and methods described in this application include viscosity modifiers. The aqueous solution may be sterilized using techniques known in the art.

A pharmaceutical preparation containing an anti-CD45 antibody or a conjugate thereof (eg, the ADC described in this application) is mixed with such an antibody or ADC with one or more optional pharmaceutically acceptable carriers. By (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), it is prepared in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dose and concentration used, including, but not limited to, phosphates, citrus. Buffers such as acid salts and other organic acids; antioxidants containing ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalconium chloride; benzethonium chloride; phenol, butyl or benzyl Alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptide; serum albumin, gelatin, Or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars such as glucose, mannose, or dextrin. Chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or polyethylene glycol (PEG) and the like. Non-ionic surfactant.

The antibodies, antigen-binding fragments, or ADCs described in this application may be administered by various routes such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, or parenteral. In any given case, the most suitable route of administration is the particular antibody or antigen-binding fragment to be administered, the patient, the pharmaceutical formulation method, the method of administration (eg, time and route of administration), the age and weight of the patient. Depends on the sex, the severity of the disease to be treated, the patient's diet, and the patient's excretion rate.

Effective doses of the antibodies, antigen-binding fragments thereof, or ADCs described in this application are, for example, from about 0.001 to about 100 mg / kg in body weight per single (eg, bolus), multiple, or continuous dose. The antibody, its antigen-binding fragment, or ADC may have an optimal blood concentration (eg, a blood concentration of about 0.0001 to about 5000 μg / mL). The above doses may be given to subjects suffering from cancer, autoimmune disease, or subjects receiving conditioning therapy in preparation for hematopoietic stem cell transplantation (eg, humans) for 1 day, 1 week, or 1 month. It may be administered more than once (for example, 2 to 10 times).

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.1 mg / kg. ~ About 0.3 mg / kg.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.15 mg / kg. ~ About 0.3 mg / kg.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.15 mg / kg. ~ Approximately 0.25 mg / kg.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.2 mg / kg. ~ About 0.3 mg / kg.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.25 mg / kg. ~ About 0.3 mg / kg.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.1 mg / kg. Is.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.2 mg / kg. Is.

In certain embodiments, the dose of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC (eg, anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient is approximately 0.3 mg / kg. Is.

In certain embodiments, the dose of the anti-CD45 antibody, an antigen-binding fragment thereof, or ADC (eg, an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to said human patient. Approximately 0.001 mg / kg to 10 mg / kg, Approximately 0.01 mg / kg to 9.5 mg / kg, Approximately 0.1 mg / kg to 9 mg / kg, Approximately 0.1 mg / kg to 8.5 mg / kg, Approximately 0.1 mg / kg 8 mg / kg, approx. 0.1 mg / kg to 7.5 mg / kg, approx. 0.1 mg / kg to 7 mg / kg, approx. 0.1 mg / kg to 6.5 mg / kg, approx. 0.1 mg / kg to 6 mg / kg, approx. 0.1 mg / kg to 5.5 mg / kg, about 0.1 mg / kg to 5 mg / kg, about 0.1 mg / kg to 4.5 mg / kg, about 0.1 mg / kg to 4 mg / kg, about 0.5 mg / kg to 3.5 mg / kg, approx. 0.5 mg / kg to 3 mg / kg, approx. 1 mg / kg to 10 mg / kg, approx. 1 mg / kg to 9 mg / kg, approx. 1 mg / kg to 8 mg / kg, approx. 1 mg / kg-7 mg / kg, approx. 1 mg / kg-6 mg / kg, approx. 1 mg / kg-5 mg / kg, approx. 1 mg / kg-4 mg / kg, or approx. 1 mg / kg-3 mg / kg.

In certain embodiments, the anti-CD45 antibody, antigen-binding fragment thereof, or ADC according to the present application administered to a human patient for treatment or conditioning is 24 hours or less, 22 hours or less, 20 hours or less. , 18 hours or less, 16 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, or 5 hours or less Has a half-life. In certain embodiments, the half-life of the anti-CD45 antibody, its antigen-binding fragment, or ADC is 5 hours to 7 hours; 5 hours to 9 hours; 15 hours to 11 hours; 5 hours to 13 hours; 5 hours to. 15 hours; 5 hours to 20 hours; 5 hours to 24 hours; 7 hours to 24 hours; 9 hours to 24 hours; 11 hours to 24 hours; 12 hours to 22 hours; 10 hours to 20 hours; 8 hours to 18 hours ; Or 14 to 24 hours.

In certain embodiments, the methods disclosed in this application minimize hepatotoxicity in patients receiving anti-CD45 antibodies, antigen-binding fragments thereof, or ADCs for conditioning. For example, in certain embodiments, by the methods disclosed in this application, liver marker levels are below known toxicity levels in said patients for more than 24 hours, 48 hours, 72 hours, or 96 hours. Will stay. In another embodiment, the method disclosed in this application ensures that the marker level of the liver remains within the reference range in said patient for more than 24 hours, 48 hours, 72 hours, or 96 hours. In certain embodiments, by the methods disclosed in this application, elevated liver marker levels are greater than 1.5-fold above the reference range, exceeding 24 hours, 48 hours, 72 hours, or 96 hours. It must not be greater than 3 times greater than the reference range, greater than 5 times greater than the reference range, or greater than 10 times greater than the reference range. Examples of liver markers that may be used in toxicity tests include alanine aminotransaminase (ALT), lactate dehydrogenase (LDH), and aspartate aminotransaminase (AST). In certain embodiments, by administering an ADC as described in this application (ie, administering in two doses instead of a single dose), liver markers (eg, AST, LDH, and / or). ALT) increases transiently. In some cases, toxic liver markers may reach high levels, but for a specific period of time (eg, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about). 72 hours, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, or less than a week) Within, the marker level of the liver returns to normal levels not associated with hepatotoxicity. For example, in humans (the average adult male), the normal non-toxic level of ALT is 7-55 units (U / L) per liter; the normal non-toxic level of AST is 8-48 U / L. Is. In certain embodiments, at least one of a patient's blood AST, ALT, or LDH levels is given to the patient a first dose of ADC and the first dose14. The toxicity level is not reached within a day. For example, the first dose is administered to the patient, followed by, for example, within 5, 10, or 14 days after the first dose is administered, the second dose, the third dose, the fourth dose. Dose or higher doses may be given. However, at least one of the blood AST, ALT, or LDH levels of the patient is between the administration of the first dose of ADC and 14 days after administration of the first dose to the patient. In addition, the toxicity level is not reached.

In certain embodiments, at least one of the patient's blood AST, ALT, or LDH levels does not rise above normal levels, does not rise above normal levels by more than 1.5-fold, and has 3 normal levels. Does not rise more than double, does not rise more than 5 times the normal level, or does not rise more than 10 times the normal level.

In the case of the conditioning procedure before hematopoietic stem cell transplantation, the antibody or its antigen-binding fragment is administered, for example, a transplant of extrinsic hematopoietic stem cells at a timing at which engraftment of extrinsic hematopoietic stem cells is optimally promoted. About 1 hour to about 1 week (for example, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours , About 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about It may be administered to the patient 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days) or more. The scope including the numbers listed in this application is also included in the intended method.

Using the methods disclosed in this application, one of ordinary skill in the art can bind an ADC, antibody, or antigen thereof capable of binding to CD45 expressed by hematopoietic stem cells to a human patient in need of hematopoietic stem cell transplantation therapy. Fragments may be administered. In this mode, the population of endogenous hematopoietic stem cells may be reduced so that engraftment of the hematopoietic stem cell transplant is promoted prior to administration of the extrinsic hematopoietic stem cell transplant. The antibody may be covalently conjugated to a toxin such as a cytotoxic molecule described in this application or known in the art. For example, an anti-CD45 antibody, or an antigen-binding fragment thereof, can be used as a cytotoxin (eg, pseudomonas extratoxin A, de bouganine, diphtheria toxin, amatoxin, such as γ-amanitin, α-amanitin, saporin, maytancin, maytancinoid, auri). Covalently associated with statins, anthracyclins, calikeamycin, irinotecan, SN-38, duocarmycin, pyrorobenzodiazepine, pyrorobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer, or variants thereof. May be conjugated to. This conjugate may be practiced using a covalent bond forming technique described in this application or known in the art. The antibody, its antigen-binding fragment, or drug-antibody conjugate is then administered, eg, intravenously, before transplanting the exogenous hematopoietic stem cells (eg, autologous, allogeneic, or allogeneic hematopoietic stem cells, etc.) into the patient. It may be administered to the patient by internal administration.

The anti-CD45 antibody, its antigen-binding fragment, or ADC, prior to hematopoietic stem cell transplantation therapy, the amount of endogenous hematopoietic stem cells, eg, about 10%, about 20%, about 30%, about 40%. It may be administered in an amount sufficient to reduce about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more. Using conventional techniques known in the art, such as FACS analysis of cells expressing characteristic hematopoietic stem cell surface antigens in blood samples taken from said patients at various intervals during conditioning therapy. , Decreased hematopoietic stem cell count may be monitored. For example, a person skilled in the art will take a blood sample from a patient at various times during conditioning therapy and perform a FACS analysis using an antibody that binds to a hematopoietic stem cell marker antigen to perform FACS analysis of the hematopoietic stem cells in the sample. Elucidation of relative concentrations may measure the extent to which endogenous hematopoietic stem cells have diminished. According to some embodiments, if the concentration of hematopoietic stem cells reaches a minimum in response to conditioning therapy with an anti-CD45 antibody, an antigen-binding fragment thereof, or an ADC, the physician said the conditioning therapy. May be terminated and preparations may be initiated for the patient in preparation for hematopoietic stem cell transplantation therapy.

The anti-CD45 antibody, antigen-binding fragment thereof, or ADC may be administered to the patient as an aqueous solution containing one or more pharmaceutically acceptable excipients (eg, viscosity modifiers). The aqueous solution may be sterilized using techniques described in this application or known in the art. For example, prior to administering the hematopoietic stem cell transplant to the patient, the antibody, an antigen-binding fragment thereof, or a drug-antibody conjugate may be applied to the patient, for example, from about 0.001 mg / kg to about 100 mg / kg. 0.001 mg / kg to about 10 mg / kg, about 0.01 mg / kg to 9.5 mg / kg, about 0.1 mg / kg to 9 mg / kg, about 0.1 mg / kg to 8.5 mg / kg, about 0.1 mg / kg 8 mg / kg, approx. 0.1 mg / kg to 7.5 mg / kg, approx. 0.1 mg / kg to 7 mg / kg, approx. 0.1 mg / kg to 6.5 mg / kg, approx. 0.1 mg / kg to 6 mg / kg, approx. 0.1 mg / kg to 5.5 mg / kg, about 0.1 mg / kg to 5 mg / kg, about 0.1 mg / kg to 4.5 mg / kg, about 0.1 mg / kg to 4 mg / kg, about 0.5 mg / kg to 3.5 mg / kg, approx. 0.5 mg / kg to 3 mg / kg, approx. 1 mg / kg to 10 mg / kg, approx. 1 mg / kg to 9 mg / kg, approx. 1 mg / kg to 8 mg / kg, approx. 1 mg / kg-7 mg / kg, approx. 1 mg / kg-6 mg / kg, approx. 1 mg / kg-5 mg / kg, approx. 1 mg / kg-4 mg / kg, or approx. 1 mg / kg- It may be administered at a dose of 3 mg / kg. At a time when extrinsic hematopoietic stem cell engraftment is optimally promoted, for example, about 1 hour to about 1 week (eg, about 1 hour, about 2 hours,) before administration of the exogenous hematopoietic stem cell transplant. About 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 Hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, About 5 days, about 6 days, or about 7 days), or earlier, the anti-CD45 antibody, its antigen-binding fragment, or ADC may be administered to the patient.

In certain embodiments, the anti-CD45 antibody, antigen-binding fragment thereof, or ADC is administered to the subject with an immunosuppressive agent. In certain embodiments, the immunosuppressive agent is an agent that reduces T cells. The immunosuppressant may be administered to the subject before, at the same time as, or after administration of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC. Exemplary immunosuppressive agents include, but are not limited to, anti-CD4 antibodies, anti-CD8 antibodies, total body irradiation, and cytoxane, and combinations thereof.

In certain embodiments, an anti-CD45 antibody, an antigen binding fragment thereof, or an ADC is administered to a subject in combination with total body irradiation (TBI) (eg, low dose TBI). In an exemplary embodiment, the subject receives a TBI at a dose of 3 Gy or less, eg, 3 Gy TBI or less, 2.5 Gy TBI or less, 2 Gy TBI or less, 1.5 Gy TBI or less, 1 Gy TBI or less, or 0.5 Gy TBI or less. ..

In certain embodiments, an anti-CD45 antibody, an antigen-binding fragment thereof, or an ADC is administered to a subject in combination with Cytoxane.

After the conditioning therapy is completed, the patient then receives an injection of extrinsic hematopoietic stem cells (eg, intravenous injection) from, for example, the same physician or another physician who performed the conditioning therapy. Sometimes. The physician may administer the patient an injection of his own, allogeneic, allogeneic hematopoietic stem cells, eg, at a dosage of 1 × 10 ³ to 1 × 10 ⁹ hematopoietic stem cells / kg. .. After administration of the implant, the doctor may, for example, by taking a blood sample from the patient and hematopoietic stem cells or cells of the hematopoietic lineage (eg, giant nuclei, platelets, platelets). Red blood cells, mast cells, myeloid blasts, basal spheres, neutrophils, eosinophils, microglia, granulocytes, monospheres, hematopoietic cells, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T- Engraftment of hematopoietic stem cell transplants may be monitored by measuring increased concentrations of lymphocytes, and B-lymphocytes, etc.). Perform this analysis, for example, 1 hour to June after hematopoietic stem cell transplantation therapy, or later (eg, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours). , About 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks , About 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about May be done 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, or later). Concentrations of hematopoietic stem cells or cells of the hematopoietic lineage increased after the transplantation therapy compared to the concentrations of the corresponding cell types prior to the transplantation therapy (eg, about 1%, about 2%, about 3%, About 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70 %, Approximately 80%, Approximately 90%, Approximately 100%, Approximately 200%, Approximately 500% Increased or Increased), use anti-CD45 antibody, or antigen-binding fragment thereof, or ADC It is shown that the treatment succeeded in promoting the engraftment of the transplanted hematopoietic stem cell transplant.

Administration of an anti-CD45 antibody, its antigen-binding fragment, or ADC can reveal engraftment of hematopoietic stem cell transplants in various experimental measurements. For example, engraftment of transplanted hematopoietic stem cells is a competitive regeneration present in the patient's bone marrow after administration of an anti-CD45 antibody, or an antigen-binding fragment thereof, followed by administration of a transplant of hematopoietic stem cells. It can be evaluated by assessing the amount of competitive repopulating unit (CRU). Furthermore, a reporter gene such as an enzyme that catalyzes a chemical reaction that produces a fluorescent product, a color-developing product, or a luminescent product is incorporated into a vector that transfects a donor's hematopoietic stem cell, and subsequently, a corresponding signal is transmitted to the bone marrow or the like. By monitoring the hematopoietic stem cells in the homing tissue, it is possible to observe the engraftment of the hematopoietic stem cell transplants. Also, by assessing the amount and viability of hematopoietic stem cells and hematopoietic progenitor cells, for example, as measured by fluorescence activated cell sorting (FACS) analysis methods known in the art. , Hematopoietic stem cells can also be observed to engraft. Engraftment is also measured during the post-transplant period by measuring the number of leukocyte cells in peripheral blood and / or by measuring the recovery of bone marrow cells by donor cells in a bone marrow aspiration sample. You can also do it.

The following examples are presented to explain to those of skill in the art as to how the compositions and methods described in this application may be used, prepared and evaluated. The following examples are purely exemplary of the invention and are not intended to limit the scope of what the inventors consider to be inventions.

Example 1. Preparation of anti-CD45 monoclonal antibody Human CD45 (specifically, the extracellular region of human CD45 RO) was used to immunize mice to obtain anti-human CD45 antibody. Cell lines expressing human CD45RO, human full-length CD45 (RABC), cynomolgus monkey RABC and mouse CD45 RABC were generated and the binding of each clone was evaluated by flow cytometry. 216 different antibodies, with primary antibodies of 10, 100, and 1000 pM, in the presence of anti-rat Fab-saporin (10 nM for cell line, 20 nM for CD34), 2 cell lines and human CD34 bone marrow. Due to the damage in the secondary Fab-saporin experiment on cells, it was evaluated in duplicate. Staurosporine was used as a 100% death control on each plate. Rat anti-human CD45 mAbs were included for comparison. Cell lines were incubated for 72 hours, CD34 cells were incubated for 120 hours, and then survival was assessed using Cell titer glo. Dose titration injury curves were then created and clones in REH cell lines and human bone marrow CD34 cells were selected.

An octet binding experiment was performed to identify mAbs with excellent cross-reactivity. Clones were selected based on their ability of the antibody to bind to human CD45RO and CD45RABC, as well as the non-human primate cynomolgus monkey CD45RABC. Five clones with cross-reactivity with human and cynomolgus monkey CD45 were identified, of which only AbA, AbB, and AbC were identified as having excellent cross-reactivity with human and non-human primate CD45 RABC.

These monoclonal antibodies were cloned and their variable regions were cloned into a vector containing human IgG1. Subsequently, the chimeric antibodies AbA, AbB, and AbC were expressed as IgG1 antibodies and their binding and activity to human CD45 cells were evaluated. Table 2 shows the degree of monovalent binding of AbA, AbB and AbC to human CD45 RABC, cynomolgus monkey CD45 RABC and rhesus monkey CD45 RABC when evaluated by octet. AbA, AbB, and AbC binding was also evaluated by flow cytometry for 24-hour PBMC binding. In each case, each clone was detected as a binding to human and cynomolgus monkey PBMC.

These antibodies were also evaluated as ADCs to determine if they were incorporated into cells expressing CD45. AbA, AbB, and AbC were each taken up into cells.

Fc variants of AbA, AbB, and AbC have also been created. Specifically, human IgG1 chimeras of AbA, AbB, and AbC were created with or without the Fc mutations D265C and V205C (for the purpose of conjugation to be ADCs). Additional Fc variant antibodies were made as having or not having H435A or I253A / H310A / H435A, and the antibodies or ADCs with further different half-lives were provided.

The amino acid sequences of the CDRs and variable regions of AbA, AbB, and AbC are shown in Table 3.

Example 2. Non-genetic toxic conditioning with amatoxin antibody drug conjugates targeting CD45 has the effect of reducing hematopoietic stem cells and immune cells in humans and non-human primates.
Preface Bone marrow transplantation (BMT) is a treatment that has the potential to cure malignant and non-malignant blood disorders. Current regimens for preparing or conditioning patients prior to BMT include regimen-related mortality and morbidity, including risk of organ toxicity, infertility and secondary malignancies. Therefore, it is a limitation of using this healing procedure. To safely condition patients for BMT, antibody drug conjugates (ADCs) targeting CD45 have been developed (targets expressed throughout the hematopoietic system, resulting in hematopoietic stem cells (HSC) prior to transplantation. ) And specifically reduce both immune cells).

Results Anti-CD45 ADCs were created by conjugating the anti-CD45 antibody AbA (chimeric AbA as IgG1) to the amatoxin (AM) payload (RNA polymerase II inhibitor). The resulting anti-CD45 AM ADC (CD45-AM) potentiates primary human bone marrow CD34 + CD90 + HSC (IC50 67 pM, FIG. 1A) and PBMC immune cells (IC50 55 pM, FIG. 1B) in vitro. Was injured. The CD45-AM ADC was then tested in vivo in humanized NSG mice. A single dose of 3 mg / kg CD45-AM reduced> 95% of human CD34 + cells in bone marrow (Fig. 1C; 14 days after dosing), and human B-, T- and bone marrow cells in the periphery and bone marrow. > 95% was reduced (Fig. 1D). A single dose of 3 mg / kg CD45-AM also reduced> 99% of peripheral human CD45 cells (Fig. 1E; 14 days after dosing; N = 3 / group). Toxin-free control non-targeting isotype-matched-ADC and anti-CD45 antibodies had minimal effect on HSCs, immune cells, or peripheral human CD45 cells. Since CD45 is highly expressed in many leukemias and lymphomas, we also evaluated the leukemia-reducing activity of CD45-AM in the ALL and 3 PDX-AML models. A single dose of 1 mg / kg CD45-AM more than doubled median survival in all four models (p-value <0.001), and some mice were free-of-disease. Was alive.

In BMT, the ADC needs to be cleared quickly so as not to reduce the incoming grafts, so a fast half-life CD45, which is t _1/2 for 8-15 hours in mice and 9 hours in cynomolgus monkeys. -Created an AM variant. In cynomolgus monkeys, increasing the dose of CD45-AM, which is cross-reactive and has a fast half-life, resulted in a dose-dependent decrease in peripheral lymphocytes. A single dose of 1 mg / kg is well tolerated, resulting in a> 95% reduction in HSC (CD34 + CD90 + CD45RA-cells) and lymphocytes in the bone marrow 14 days after dosing. > 80% reduction (Fig. 1D).

In conclusion, targeting CD45 using ADCs with amatoxin resulted in a strong reduction of human HSCs and immune cells in vitro and in vivo. The anti-CD45 amatoxin ADC significantly reduced the disease burden in multiple leukemia models. Preliminary assessments of fast half-life ADCs in cynomolgus monkeys showed an efficient reduction of HSCs and immune cells in the bone marrow. Amatoxin ADCs targeting CD45 are i) non-genetic toxicity; ii) amanitin has low cell permeability as a free toxin, thus avoiding bystander toxicity; and iii) cell cycle. It may damage cells that are orbiting and cells that are not orbiting the cell cycle, the latter being essential for the effective elimination of HSCs and immune cells. By combining these characteristics, it is possible to perform more safe and targeted conditioning, and to expand the use of BMT in malignant and non-malignant symptoms.

Example 3. Conditioning agents that are anti-CD45 antibody drug conjugates (ADCs) are refractory or high risk for allogeneic hematopoietic stem cell transplantation with in vivo anti-leukemia activity in cell line and patient-derived xenograft models. This is a potentially curative approach in patients with hematopoietic malignancies. Prior to transplantation, patients are conditioned by high-dose chemotherapy, or chemotherapy and total body irradiation, which are a substantial risk of early and late morbidity and death. is connected with. As a result, many eligible patients do not consider transplantation, and 2/3 of the transplanted patients are shinobi only for reduced intensity conditioning associated with increased relapse rates. It may be a patient. Therefore, there is an urgent need for safer and more effective conditioning agents with improved disease control. To meet this need, the antibody AbB conjugated to amatoxin (AM), which targets CD45 (Rahul 2018) expressed in all lymphoid hematopoietic cells and almost all hematological malignancies, except multiple myeloma. We have developed a novel antibody drug conjugate (ADC) using chimeric AbB) in human IgG1. The purpose of this project was to measure the anti-leukemic ability of anti-CD45-AM, a drug that has already been shown to reduce primary human HSPC in vitro and in vivo.

Method. ADC was expressed in REH-Luc (an AML cell line labeled with luciferase, expressing CD45), as well as both CD117 and CD45, with different proliferative kinetics (median survival time of vehicle-treated groups). 43, 63, 82 days post-inoculation), patient-derived xenografts (PDX) (J000106132, J000106565, J000106134) (J000106132, J000106565, J000106134), prepared with FLT-3 + NPM1 + AML samples. Tested at Jackson Laboratories).

result. In the REH-Luc model, a single injection of 1 mg / kg CD45-AM on day 5 after AML inoculation was 15 days compared to vehicle-treated control or unconjugated anti-CD45 antibody. At median, they survived longer (n = 10 mice / group, p <0.0001). Using a 4-5 mouse / group / AML-PDX model, survival was significantly increased at 0.1 mg / kg CD45-AM recipients compared to vehicle controls.

Conclusion. CD45-AM is a potent anti-leukemia agent based on these data in a humanized mouse model with established AML, in addition to reducing normal host HSPC. Along with previous reports on the efficacy of CD45-AM conditioning agents, these non-genetic ADCs are recipients of patients with active disease and dose-reduced conditioning with a high risk of disease relapse. may be useful in recipient).

Example 4. A single dose of an antibody drug conjugate (ADC) targeting CD45 has potent in vivo anti-leukemia activity and is a life-prolonging allogeneic myeloid transplant in a patient-derived AML model. (BMT) is a potentially curative approach in patients with refractory or high-risk hematological malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Prior to transplantation, prepare the patient for non-specific, high-dose chemotherapy alone or in combination with total body irradiation, but these are early and late pathological It is associated with the condition (eg, organ toxicity, infertility, secondary malignancies, etc.), as well as the substantial risk of death. As a result, many eligible patients did not consider transplantation, and 2/3 of the transplanted patients were only for conditioning with reduced intensity associated with increased relapse rates. It can be tolerable (Scott et al. Journal of Clinical Oncology 2017, 1154-1161). Therefore, there is an urgent need for safer and more effective conditioning agents with improved disease control. To meet this need, hematopoietic stem and progenitor cells and AML and MDS cells in approximately 80% of patients (Gao et al. PLOS One. 2015) CD117 (C-KIT, Pearse 2018), and Two novel antibody drug conjugates targeted to CD45 (Palchaudhuri 2018) expressed in all lymphohematopoietic cells and almost all hematopoietic tumors except multiple myeloma, and conjugated to amatoxin (AM) ADC), we have developed. The purpose of this project is to design a non-genetically toxic drug with the dual benefit of reducing primary human hematopoietic stem progenitor cells (HSPCs) while reducing the disease burden in the leukemia model. there were.

METHODS : The anti-CD45 antibody used in the ADCs in the examples below is the anti-CD45 antibody AbB, which has a drug-to-antibody ratio (DAR) of 4 and is (interchain) conjugated to amanitin. ADC expresses CD45-expressing ALL cell lines (5x10 ⁶ cells / mouse), CD45-labeled human leukemia cells derived from immortalized cell lines (REH-Luc, luciferase), and CD45, with different proliferative kinetics. (The median survival time of the vehicle-treated group is 43, 63, 82 days post-inoculation), xenografts from three patients (patient-) prepared with FLT-3 + NPM1 + AML samples. Derived xenografts (PDX)) [AML # 1 (J000106132), AML # 2 (J000106565), AML # 3 (J000106134)] (Jackson Laboratories), evaluated in xenograft mouse models. All in vivo studies are institutionalized according to the Guide for the Care and Use of Laboratory Animals published by the National Research Council of the National Academies. Conducted with the approval of the Animal Care and Use Committee.

result
Effective Target Reduction in Vitro Human PBMCs to anti-CD45-amatoxin conjugates (antibodies AbB-amatoxin; "CD45-AM") or amatoxins for in vitro injury assays using human PBMCs. Cell viability was measured as luminescence (RLU; y-axis) by Celltiter Glo while culturing for 4 days in the presence of conjugated IgG1 isotype (“isotype-AM”) and varying antibody concentration (x-axis). (Fig. 2A). Primary human CD34 ⁺ bone marrow cells with CD45-AM or isotype-AM for 5 days for in vitro injury assay using human HSCs (ie, bone marrow cells selected in isolated primary human CD34 + (BMC)). The counts (y-axis) of cultured and live CD34 + CD90 + HSCs were determined by flow cytometry with varying antibody concentrations (x-axis).

The results of FIGS. 2A and 2B show that the CD45-ADC in vitro damages a CD45-expressing cell line (eg, PBMC; 614 pM; FIG. 2A) or a primary human CD34 ⁺ CD90 + HSC (68 pM, FIG. 2B). Shows that it is very effective.

In the REH-luciferase ALL xenograft model, anti-CD45-AM doubles median survival up to 40 days.
Heterologous transplanted mice inoculated with human leukemia cells derived from immortalized cell lines expressing CD45 (REH-Luc, ALL cell line expressing CD45 (5 × 10 ⁶ cells / mouse) labeled with luciferase). The CD45-ADC was tested in the model. As shown in FIG. 3A, a single injection of 1 mg / kg anti-CD45-AM on day 5 after ALL inoculation with vehicle (PBS) treated control or unconjugated anti-CD45 antibody. By comparison, median survival was longer for 15 days (n = 10 mice / group, p <0.0001). In addition, pseudocolor images of typical bioluminescent signals were obtained 19 days after transplantation for anti-CD45-AM and isotype-AM treated groups using the IVIS Imaging System (Perkin Elmer) (Perkin Elmer). FIG. 3B). These results indicate that in the REH-Luc xenograft model, anti-CD45-AM doubles median survival up to 40 days.

In three patient-derived AML models, anti-CD45-AM and anti-CD117-AM prolong survival 2- to> 4-fold, then express CD45 and differ in proliferative kinetics (in the medium). Median survival in the treated group is 43, 63, 82 days post-inoculation), patient-derived xenografts (patient-derived xenografts) from three patients prepared with FLT-3 + NPM1 + AML samples. PDX)) (Jackson Laboratories), CD45-ADC was tested in [AML # 1 (J000106132), AML # 2 (J000106565), AML # 3 (J000106134)]. Table 4 summarizes the characterization of the AML PDX model.

For 3 PDXs, 4-5 mice / group / AML-PDX model with 2-5% precursor cells in blood (ie, 2-5% tumors in peripheral blood) ADC (CD45-AM, isotype-AM (ISO-AM) or vehicle PBS) was administered to AML-PDX mice in a single intravenous dose. 4A and 4H show the results of a flow cytometry assay to assess the cell surface expression of CD45 on CD33 + spleen cells derived from diseased mice. As shown in FIG. 4B-4D, survival was significantly increased at 1 mg / kg CD45-AM recipient compared to medium or isotype controls (Table 5; FIG. 4B-4D). ). 4E-4G correspond to the same data shown in FIGS. 4B-4D, but each show a shorter post-transplant duration, 1 mg / kg CD45-Am (or isotype-Am control,). Or the timing of administration of PBS medium control), and the survival curve of anti-CD45-Am and control (ie, isotype-Am and / or PBS medium control) at 50% survival of each PDX AML model. Shows the difference in days between and (ie PDX AML # 1 (106 days), PDX AML # 2 (81 days), and PDX AML # 3 (> 150 days)).

These results indicate that a single dose of anti-CD45-AM is well tolerated and that it strongly damages human CD34 + CD90 + hematopoietic stem cells, AML cell lines, and human PBMCs in vitro. It was proved that it can be done. Furthermore, based on these data in humanized mouse models with established AML, a single dose of anti-CD45-AM prolongs the survival of the established leukemia model (cell line and patient origin). And a strong anti-leukemia effect appears.

Non-genetically toxic ADCs can be used to reduce the burden of the disease in patients with active disease and in weight-loss conditioning recipients who are at high risk of relapse. can.

Example 5. Targeting CD45 with amanitin antibody-drug conjugates is aimed at transplant conditioning and effectively reduces human HSCs and immune cells Bone Marrow Transplantation (BMT) cures malignant and non-malignant blood disorders It is a potential treatment and has shown remarkable results in autoimmune diseases. Prior to BMT, patients are prepared with high-dose chemotherapy alone or with total body irradiation, both in early and late morbidity (eg, organ toxicity, infertility). Diseases, secondary malignancies, etc.), as well as a substantial risk of death. This greatly limits the use of BMT in malignant and non-malignant conditions. To address these issues, we have developed a non-genetically toxic antibody drug conjugate (ADC) that safely conditions patients for BMT, targets hematopoietic stem cells (HSCs) and immune cells. are doing.

Alpha-amanitine ADCs targeting to CD45 may be suitable for conditioning patients for BMT, because it is 1) non-genetic toxicity; 2) amanitin as a free toxin. Poor cell permeability avoids bystander toxicity; 3) damages cells that are or do not cycle, the latter effectively removing HSCs and immune cells. This is because there are things that are essential for making them do. Elimination of both immune cells and HSCs using an anti-CD45 ADC may allow allogeneic transplantation in malignant and non-malignant settings. Furthermore, this measure is also considered to be effective in enabling immune reset via autologous transplantation in patients with autoimmune diseases.

Method
CD45-ADC
CD45-AM is an antibody consisting of a chimeric human anti-CD45 antibody conjugated to amatoxin (AM), an RNA polymerase II inhibitor that can reduce cells that are or are not in the cell cycle. -Drug conjugate. The isotype control ADC is a non-targeting monoclonal human IgG antibody conjugated to amatoxin.

In vitro cell culture Human peripheral blood mononuclear cells (PBMC) were cultured in RPMI 1640 containing 10% FBS for 4 days in the presence of ADC and viability was assessed by CellTiter Glo. Human HSCs (BM cells selected with CD34 +) were cultured in SFEM medium containing IL-6, TPO, FLT-3 ligand and SCF. Five days after incubation with ADC, surviving HSCs were quantified by flow cytometry.

In vivo studies Humanized NSG mice (before transplanting human cord blood CD34 + cells) were purchased from Jackson Laboratories. Mice were injected with a single intravenous injection of each antibody-drug conjugate. Male cynomolgus monkeys were given a single intravenous dose over 1 hour. All in vivo studies are institutionalized according to the Guide for the Care and Use of Laboratory Animals published by the National Research Council of the National Academies. Conducted with the approval of the Animal Care and Use Committee.

result
Efficacy in humanized NSG mice An anti-CD45 ADC was generated by conjugating the anti-CD45 antibody AbB to an amatoxin (AM) payload (RNA polymerase II inhibitor) (DAR4; interchain conjugate). .. We investigated the CD45-AM ADC in vivo in humanized NSG mice. As shown in the schematic of FIG. 5A, CD45-AM or control antibody was administered as a single dose on day 0. Peripheral PBMCs were collected on days 7 and 14, and human CD45 cells were examined by flow cytometry. On day 14, the presence or absence of human CD34 ⁺ cells in the bone marrow was quantified.

As shown in FIGS. 5B and 5C, CD45-AM ADC selectively reduces human HSCs and immune cells in humanized NSG mice. FIG. 5B shows an anti-CD45-amatoxin (“CD45-AM”), an unconjugated anti-CD45 antibody (“CD45-naked”), or a control non-targeting isotype-matched ADC ( The results of an assay showing the absolute number of human CD45 + cells in peripheral blood 14 days after injection of "isotype-AM") are shown. FIG. 5C shows the absolute number of human CD34 ⁺ cells in the bone marrow of humanized NSG mice 14 days after administration. Similar results were obtained for CD34 ⁺ CD90 ⁺ (data not shown). These results indicate that CD45-AM ADC selectively reduces human HSCs and immune cells in humanized NSG mice.

PK and efficacy in primates
Since BMT may require rapid clearance of the ADC to avoid reducing incoming implants, we have 8-15 hours in mice and in cynomolgus monkeys. Created a CD45-AM variant with a fast half-life of t _1/2 for 9 hours (conjugated to amatoxin (AM) with a drug to antibody ratio (DAR) of 2). Also, AbA with Fc variants D265C and H435A (EU index) is used).
As shown in FIG. 6A, male crab monkeys received a single intravenous dose of the fast half-life CD45-AM variant at 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg over 1 hour. (N = 3 / group, except for n = 2 for 1 mg / kg).

FIG. 6B and Table 6 show the results of an in vivo pharmacokinetic assay showing that the same fast half-life CD45-AM ADC has a fast half-life PK profile in cynomolgus monkeys. As shown in FIG. 6B, CD45-AM exhibits a target-mediated drug disposition (TMDD) PK, which exhibits a non-linear PK and has a short half-life suitable for transplantation. show. As used in FIG. 6B, "TAb" refers to all antibodies used to detect anti-hIgG1, while ADC refers to detecting anti-amanitin. These data also showed that the ADC is stable in vivo because the Tab level overlaps with the ADC level.

FIG. 6C represents the same data in FIG. 6B, but over a longer period of time after administration, further providing a window for injecting the implant. As shown in FIG. 6C, the window is more than 4 days after dose administration.

Next, the CBC evaluated that peripheral lymphocytes were reduced by the same fast half-life CD45-AM variant (ie, amanitin-conjugated AbA with variant Fc mutants D265C and H435A (EU index)). It is shown in FIG. 7A. Furthermore, 14 days after administration of the CD45-AM variant with the same fast half-life, bone marrow HSC (CD34 + CD90 + cells), T cells and B cells were observed to decrease in a dose-dependent manner (FIG. 7B). The results in FIG. 7C (data from the time course shown in FIG. 7A, specifically representing data 1 day after dosing) show a 80% reduction in peripheral lymphocytes at 0.3 mg / kg and 1 mg / kg doses. Show that. The results in FIGS. 7A-7C show that a fast half-life CD45-AM ADC effectively reduces CD45 cells in cynomolgus monkeys. ALT, bilirubin, and platelets were within the normal range after administration of CD45-AM.

In conclusion, these studies show that a single dose of CD45-AM ADC is well tolerated and can reduce human immune cells and CD34 ⁺ cells in the bone marrow of humanized NSG mice. Was demonstrated. Furthermore, it was demonstrated that a single dose of CD45-AM ADC with a fast half-life is well tolerated and can reduce immune cells and CD34 + cells in the bone marrow of cynomolgus monkeys. These data also show a decrease in HSC (CD34 + CD90 + CD45RA-) and immune cells in the bone marrow on day 14. No reduction was observed with isotype-amanitin-conjugated or unconjugated antibodies.

Non-genetic toxic conditioning with CD45-ADC provides an approach for safer conditioning prior to BMT and may significantly increase the number of patients eligible for transplantation. There is. In addition to its use in transplantation for malignant diseases, this approach may make it possible to prepare patients for BMT in non-malignant conditions (eg, autoimmune diseases, etc.).

Example 6. Analysis of red blood cell count, platelet count, plasma ALT and bilirubin in an in vivo dose escalation study in a cohort of cynomolgus monkeys (3 monkeys per cohort) at various doses (ie, 0.1 mg / kg) on day 0 Anti-CD45-amanitin ADC (conjugated AbA, Fc mutants D265C and H435A (EU index)) with fast half-life at various doses at 0.3 mg / kg; 1 mg / kg; or control (PBS)). Has) was administered. Blood was collected throughout the study. Hematological examinations were performed throughout the study. Red blood cell count (10 ⁶ / μL), platelet count (10 ³ / μL), plasma level of ALT (alanine aminotransaminase), and plasma level of bilirubin were measured and shown in FIGS. 8A-8D, respectively. , The number of days after administration is shown in a graph on the horizontal axis.

The results in FIGS. 8A and 8B show that administration of a fast half-life anti-CD45-amanitin ADC produced significant changes in erythrocyte or platelet levels compared to PBS control, even at the highest dose of ADC. Not shown. These data also showed that administration of a fast half-life anti-CD45-amanitin ADC did not cause anemia or thrombocytopenia in non-human primates.

The results in FIGS. 8C and 8D show that administration of a fast half-life anti-CD45-amanitin ADC shows no significant change in plasma levels of ALT and bilirubin compared to PBS control, even at the highest dose of ADC. I showed that. In addition, the following additional parameters (ie, GGT, albumin, BUN, ALP, creatinine, or glucose) were not changed beyond the upper limit normal (ULN) (data not shown). ). Furthermore, no change in food intake or body weight was observed. Taken together, these data show that administration of a fast half-life anti-CD45-amanitin ADC does not result in elevated liver enzymes or changes in liver function (PT, albumin), and the ADC does not. It has been shown to be safe and well tolerated in human primates.

Example 7. Epitope mapping
The epitope to which AbA binds was mapped using cross-linking mass spectrometry. Cross-linking experiments allow direct analysis of non-covalent interactions by High-Mass MALDI mass spectrometry. By mixing protein samples with non-covalent interactions with a specially developed cross-linking mixture (Bich, C et al. Anal. Chem., 2010, 82 (1), pp 172-179). , Non-covalent complexes can be detected with high sensitivity and specificity. The covalent bonds generated allow the interacting species to withstand the sample preparation process and MALDI ionization. A special High-Mass detection system makes it possible to characterize interactions in the High-Mass range.

To determine the epitope of AbA with high resolution, the protein complex was incubated with a deuterated cross-linker and subjected to multiple multi-enzymatic cleavage. The protein complex was an AbA variant antibody (having the same epitope as AbA) bound to human CD45. After enriching the cross-linked peptide, the sample was analyzed by high resolution mass spectrometry (nLC-LTQ-Orbitrap MS) and the generated data was analyzed using XQuest and Stavrox software.

After proteolysis of the dehydrogenated d0d12 protein complex CD45 / AbA with trypsin, chymotrypsin, ASP-N, elastase and thermolysin, nLC-orbitrap MS / MS analysis revealed that the AbA epitope was a peptide. RNGPHERYHLEVEAGNT (SEQ ID NO: 38) contains residues, in particular SEQ ID NO: 37 (fragment of CD45 isoform corresponding to NP_002829.3) 486R, 493Y and 502T. It has been shown to interact with amino acids on human CD45 corresponding to. These results are shown in FIGS. 9A and 9B. FIG. 9A describes an amino acid fragment containing an epitope of AbA (SEQ ID NO: 39), in particular the region RNGPHERYHLEVEAGNT (SEQ ID NO: 38). CD45 contains a mucin-like domain, a d1-d4 fibronectin-like domain, and a transmembrane domain and a phosphatase domain. Based on this result, AbA interacts with the fibronectin d4 domain of CD45, which is conserved among alternative spliced CD45 isoforms [eg, RO, RA, RB, RC, RABC, etc.]. It works. As shown in FIG. 10, the d1-d4 fibronectin-like domain of human CD45R0 (SEQ ID NO: 40) is 68% of the corresponding region of cynomolgus monkey CD45 (SEQ ID NO: 41). It has identity and means that AbA binds to both cynomolgus monkey CD45 and human CD45.

Other Embodiments All publications, patents, and patent applications referred to in this application are to the same extent as if each independent publication or patent application was specifically and individually presented and incorporated by reference. Incorporated by reference in this application.

The invention has been described in the context of its particular embodiment, but further modifications are possible, and the present application is generally any modification of the invention in accordance with the principles of the invention. , Use, or indications, as well as deviations from the invention that fall within the known or customary practice within the art in which the invention relates, and the essential features described in this application and subject to the claims. It will be appreciated that it is intended to include any modification, use, or adaptation of the invention, including any deviations from the invention that may apply to.

Other embodiments are within the scope of the claims of the present application.

Claims (29)

An isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising:
(i) CDR1 having the amino acid sequence set forth in SEQ ID NO: 2, CDR2 having the amino acid sequence set forth in SEQ ID NO: 3, and SEQ ID NO: set forth in 4 and CDR1 having the amino acid sequence set forth in SEQ ID NO:6, the amino acid sequence set forth in SEQ ID NO:7 CDR3 having the amino acid sequence set forth in SEQ ID NO:8;
(ii) CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and SEQ ID NO: set forth in 12 and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15. CDR3 having the amino acid sequence set forth in SEQ ID NO: 16; or
(iii) CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and SEQ ID NO: set forth in 20 and a CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, the amino acid sequence set forth in SEQ ID NO: 23. CDR3 having the amino acid sequence set forth in SEQ ID NO:24. An anti-CD45 antibody, or antigen-binding portion thereof, according to claim 1, wherein:
(i) its heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1;
(ii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9; or
(iii) its heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:17; An anti-CD45 antibody, or antigen-binding portion thereof, according to claim 1, wherein:
(i) its light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 5;
(ii) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13; or
(iii) its light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:21; An isolated anti-CD45 antibody, or antigen-binding portion thereof, wherein it is
(i) SEQ ID NO: specifically binds to an epitope of human CD45 comprising residues 486R, 493Y, and 502T of 37 and binds to cynomolgus and rhesus CD45;
(ii) specifically binds to an epitope of human CD45 comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38) and binds to cynomolgus and rhesus monkey CD45; or
specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 39) and binds to cynomolgus and rhesus monkey CD45; or
(iii) (a) SEQ ID NO: specifically binds to an epitope of human CD45 comprising residues 486R, 493Y, and 502T of 37;
(b) at least 1 additional amino acid, at least 2 additional amino acids, at least 3 additional amino acids, at least 4 additional amino acids in a peptide comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38) or at least 5 additional amino acids, wherein the residues of said additional amino acids are not the residues listed in (a); and
(c) binds to cynomolgus and rhesus monkey CD45; 5. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 4, wherein said antibody competes with antibody AbA for binding to human CD45. 6. The anti-CD45 antibody, or antigen-binding portion thereof, of any one of claims 1 to 5, wherein it comprises
(i) a deimmunized antibody, or antigen-binding fragment thereof;
(ii) a humanized antibody, or antigen-binding fragment thereof, or
(iii) a chimerized antibody, or antigen-binding fragment thereof;
is. 7. An isolated deimmunized monoclonal anti-CD45 antibody, or antigen-binding portion thereof, wherein it specifically binds human CD45 and cynomolgus monkey CD45, wherein any one of claims 1-6. The anti-CD45 antibody, or antigen-binding portion thereof, according to A. is the parent antibody, or antigen-binding portion thereof, of a deimmunized anti-CD45 antibody. 8. Anti-CD45 antibody according to any one of claims 1 to 7, wherein it is an intact monoclonal IgG antibody. 8. The anti-CD45 antibody of any one of claims 1-7, comprising an Fc region comprising at least one amino acid substitution that is D265/L234/L235, H435 or I235/H310/H435 (EU numbering); or an antigen-binding portion thereof. 10. The anti-CD45 antibody, or antigen-binding portion thereof, of any one of claims 1-9, wherein said antibody, or antigen-binding portion thereof, is IgG. 11. An antibody drug conjugate (ADC) comprising the antibody of any one of claims 1-10 conjugated to a cytotoxin via a linker. 12. The ADC of claim 11, wherein said cytotoxin is an RNA polymerase inhibitor. 13. The ADC of claim 12, wherein said RNA polymerase inhibitor is amatoxin. 14. The ADC of claim 13, wherein said amatoxin is represented by formula (IV)

wherein R ₁ is H, OH, OR _A , or OR _C ;
_R2 is _H , OH, ORB, or _ORC ;
R _A and R _B together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group;
R ₃ is H, _RC , or _RD ;
_R4 , _R5 , R6 _, and _R7 are each independently H, OH, _ORC , _{ORD, RC ,} or _RD ;
_R8 is _OH , _NH2 , _ORC , _ORD , _NHRC , or _NRCRD ;
_R9 is _H , OH, ORC, or _ORD ;
X is -S-, -S(O)-, or _-SO2- ;
R _C is -LZ;
_RD is optionally substituted C1 _- C6 alkyl, optionally substituted C1 _- C6 _heteroalkyl , optionally substituted _{C2 - C6} alkenyl, optionally C2 _- C6 heteroalkenyl substituted with, optionally substituted C2 _{- C6} alkynyl, optionally substituted _{C2 -} C6 _{heteroalkynyl} , optionally substituted cycloalkyl , optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;
L is optionally substituted C1 _- C6 alkylene, optionally substituted C1 _- C6 heteroalkylene, optionally substituted _{C2 - C6 alkenylene} , optionally substituted C2 _- C6 heteroalkenylene, optionally substituted C2 _- C6 _alkynylene , optionally substituted _{C2 -} C6 _{heteroalkynylene} , optionally substituted cycloalkylene , optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, peptide, dipeptide, -(C=O)-, or combinations thereof; and,
Z is a chemical substructure formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen-binding fragment, wherein Am contains exactly one R _C substituent. 12. The ADC of claim 11, wherein said ADC has the formula:

Here, Ab represents the position of binding of said anti-CD45 antibody. 12. The ADC of claim 11, wherein said ADC has the formula:

Here, Ab represents the position of binding of said anti-CD45 antibody. 13. The ADC of claim 12, wherein said RNA polymerase inhibitor is amanitin. 12. The ADC of claim 11, wherein said cytotoxin is Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38 , duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers. 19. The ADC of claim 18, wherein said auristatin is MMAE or MMAF. 20. The ADC of any one of claims 11-19, wherein said antibody is conjugated to said toxin via a cysteine residue in the Fc domain of said antibody. 21. The ADC of claim 20, wherein said cysteine residue is introduced by amino acid substitution in the Fc domain of said antibody. 22. A pharmaceutical composition comprising the antibody or ADC of any one of claims 1-21 and a pharmaceutically acceptable carrier. 22. A method of depleting a hematopoietic stem cell (HSC) population in a human patient, said method comprising administering to said patient an effective amount of the antibody or ADC of any one of claims 1-21. include. 24. The method of claim 23, further comprising administering to said patient a graft comprising hematopoietic stem cells. 25. The method of claim 24, wherein said graft is allogeneic or autologous. A method comprising administering to a human patient a graft comprising hematopoietic stem cells, wherein said patient is administered an amount sufficient to reduce a population of hematopoietic stem cells or a population of immune cells in said patient. Has previously been administered the antibody or ADC of any one of paragraphs 1-21. 27. The method of claim 26, wherein said hematopoietic stem cells are CD45+ cells, or wherein said immune cells are CD137+, CD2+ or CD5+ cells, or T cells. 28. The method of any one of claims 23-27, wherein the patient has a hematologic disease, a metabolic disorder, cancer, or an autoimmune disease or severe combined immunodeficiency disease. SCID). 22. A method of treating leukemia in a human patient, wherein said method comprises administering an antibody or ADC according to any one of claims 1-21 to a human patient with leukemia.

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