Anti-CD123 antibodies and neodegrader conjugates

Abstract

The present disclosure provides binding moieties that specifically bind to CD123 and GSPT1 neoDegrader conjugates comprising the same. Also provided are compositions comprising the binding moieties and neoDegrader conjugates. The compounds and compositions are useful for treating a disease or condition, e.g., hematological cancers.

Description

ANTI-CD123 ANTIBODIES AND NEODEGRADER CONJUGATESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 701,090, filed September 30, 2024 which is incorporated by reference herein in its entirety.DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted Sequence Listing (Name: 4547_033PC01_Sequencelisting_ST26; Size: 61,960 bytes; and Date of Creation: September 29, 2025), filed with the application, is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure provides binding moieties that specifically bind to CD 123 as well as GSPT1 neoDegrader conjugates containing the same. Also provided are compositions comprising the binding moieties and conjugates. The conjugates and compositions are useful for treating hematological cancers.BACKGROUND

[0004] Targeted protein degraders (TPDs) have expanded the breadth of therapeutic options through both their catalytic mechanism of action and ability to degrade previously “undruggable” target proteins. More recently, TPDs have been conjugated to tumor-targeting antibodies to improve efficacy and tolerability of cancer treatments. CD123 (also known as interleukin-3 receptor alpha chain) has been identified as a potential target for the treatment of hematological cancers, and monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies directed against CD 123 are under evaluation as possible anti -leukemic drugs. However, TPDs have not previously been conjugated to CD123-binding agents, and there is a continuing need for new compounds that can effectively treat hematological cancers.SUMMARY

[0005] In some aspects, the present disclosure provides antibodies and antigen-binding fragments thereof that immunospecifically bind to CD 123. In some aspects, the antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:3, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:8, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NOV, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:7. In some aspects, the antibody or antigen-binding fragment comprises a VH with an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the antibody or antigen-binding fragment comprises a VH with an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the antibody or antigen-binding fragment comprises a VH with an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the antibody or antigen-binding fragment comprises a VH with an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the antibody or antigen-binding fragment comprises a VH with an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the antibody or antigen-binding fragment comprises a VH with an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the VL comprises an amino acid that is not Y position 36 according to Kabat numbering, optionally wherein the VL comprises an F position 36 according to Kabat numbering. In some aspects, the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO:21 and / or a VL comprising the amino acid sequence of SEQ ID NO:22.

[0006] In some aspects, the antibody or antigen-binding fragment is an IgG antibody or an antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment is an IgGl antibody or antigen-binding fragment thereof. In some aspects, the antibody or antigenbinding fragment comprises (i) an N297A mutation according to EU numbering and / or (ii) a LALA (L234A and L235A) mutation according to EU numbering. In some aspects, the antibody or antigen-binding fragment thereof comprises (i) an N297A mutation according to EU numbering. In some aspects, the antibody or antigen-binding fragment comprises a constant region comprising the amino acid sequence of any one of SEQ ID NOs:47-53. In some aspects, the constant region comprises the amino acid sequence of SEQ ID NO:51. In some aspects, the antibody or antigenbinding fragment thereof comprises a constant region comprising an engineered cysteine.

[0007] In some aspects, the antibody or antigen-binding fragment comprises (i) a heavy chain comprising the amino acid sequences of SEQ ID NO:55 and / or (ii) a light chain comprising the amino acid sequence of SEQ ID NO:56.

[0008] The present disclosure also provides conjugates. In some aspects, a conjugate provided herein comprises an antibody or antigen-binding fragment provided herein conjugated to a GSPT1 neodegrader.

[0009] In some aspects, a conjugate provided herein is a conjugate of formula (I) or formula (II):(II), or a pharmaceutically acceptable salt thereof, wherein:

[0010] a is 1 to 10;

[0011] n is 0 or 1;

[0012] A is phenyl or a C4-Ciocycloalkyl ring;

[0013] U is selected from NH, O, S, and CF2;

[0014] R1is independently selected from hydrogen and halo;

[0015] R10is selected from -CH3, -C(O)R30, -N(R40)2, -(CH2)nOH, -(CH2)n’N(R40)2,-(CH2)n Q(CH2)m OH, -(CH2)n Q(CH2)m SH, and -(CH2)n’Q(CH2)mN(R40)2; wherein:

[0016] R30is hydrogen or Ci-Cealkyl;

[0017] each R40is independently hydrogen or Ci-Cealkyl;

[0018] Q is O, S, or NR40;

[0019] n’ is 1-6; and

[0020] m’ is 2-5;

[0021] R20is selected from hydrogen, -(CH2CH2O)v -CH3, C2-Cealkenyl, Ci-Cealkyl; C2-Cealkynyl, benzyl, Cs-Cecycloalkyl, and C3-C6cycloalkyl(Ci-C3alkyl), wherein v’ is from 1 to 24;

[0022] R50and R51are independently selected from hydrogen and deuterium;

[0023] X is selected from -NR200-, =C(CH3)-, -Q’-(CH2)n and -Q’(CH2)m”Q”(CH2)n”- ; wherein:

[0024] Q’ and Q” are each independently O, S, or N(R200)v, wherein:

[0025] v is 1 or 2;

[0026] each R200is independently hydrogen or Ci-Cealkyl;

[0027] n” is an integer from 1 to 6; and

[0028] m’ ’ is an integer from 2 to 6;

[0029] wherein the left side of each group is attached to L and the right side is attached to A;

[0030] provided that when X is NH or -Q’-(CH2)n ”-, R1is halo;

[0031] each Y is independently S or O;

[0032] L is a cleavable linker or non-cleavable linker;

[0033] L50is a cleavable linker;

[0034] and

[0035] Bm is an antibody or antigen-binding fragment thereof provided herein.

[0036] In some aspects, R50and R51are each hydrogen.

[0037] In some aspects, a is an integer from 1 to 10.

[0038] In some aspects, a is from 2 to 8.

[0039] In some aspects, a is an integer from 2 to 8.

[0040] In some aspects, the conjugate is a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker.

[0041] In some aspects, L is selected from the group consisting of

[0043] wherein:

[0044] p is an integer from 1 to 10;

[0045] is the point of attachment to X; and*

[0046] is the point of attachment to the binding moiety.

[0047] In some aspects, L is selected from the group consisting of

[0049] wherein:

[0050] p is an integer from 1 to 10;

[0051] X is the point of attachment to X; and*

[0052] is the point of attachment to the binding moiety.

[0053] In some aspects, L is

[0055] In some aspects, p is 5.

[0056] In some aspects, the conjugate is a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker.

[0057] In some aspects, the cleavable linker is cleavable by a protease.

[0058] In some aspects, L is selected from the group consisting of

[0060] wherein:

[0061] q is an integer from 2 to 10;

[0062] Z1, Z2, Z3, and Z4are each independently absent or a naturally-occurring amino acid residue in the L- or D-configuration, provided that at least two of Z1, Z2, Z3, and Z4are amino acid residues;

[0063] the point of attachment to X; and

[0064] is the point of attachment to the binding moiety.

[0065] In some aspects, Z1, Z2, Z3, and Z4are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D- glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D- asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; provided that at least two of Z1, Z2, Z3, and Z4are amino acid residues.

[0066] In some aspects:

[0067] Z1is absent or glycine;

[0068] Z2is absent or selected from the group consisting of L-glutamine, D-glutamine, L- glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine;

[0069] Z3is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; and

[0070] Z4is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D- citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0071] In some aspects, L is

[0073] In some aspects, q is 5.

[0074] In some aspects, L is a bioreducible linker.

[0075] In some aspects, L is selected from the group consisting of

[0076]

[0077] wherein:

[0078] q is an integer from 2 to 10;

[0079] R, R’, R”, and R’” are each independently selected from hydrogen, Ci-CealkoxyCi-Cealkyl, (Ci-Ce^NCi-Cealkyl, and Ci-Cealkyl, or, two geminal R groups, together with the carbon atom to which they are attached, can form a cyclobutyl or cyclopropyl ring;

[0080] the point of attachment to X; and

[0081] / is the point of attachment to the binding moiety.

[0082] In some aspects, L is an acid cleavable linker.

[0083] In some aspects, L is selected from the group consisting of

[0084]

[0085] wherein:

[0086] q is an integer from 2 to 10;

[0087] is the point of attachment to X; and

[0088] is the point of attachment to the binding moiety.

[0089] In some aspects, L is a click-to-release linker.

[0090] In some aspects, L is selected from

[0092] wherein:

[0093] q is an integer from 2 to 10;

[0094] is the point of attachment to X; and

[0095] r is the point of attachment to the binding moiety.

[0096] In some aspects, L is a pyrophosphatase cleavable linker.

[0097] In some aspects, L is

[0098]

[0099] wherein:

[0100] q is an integer from 2 to 10;

[0101] is the point of attachment to X; and★

[0102] / is the point of attachment to the binding moiety.

[0103] In some aspects, L is a beta-glucuronidase cleavable linker.

[0104] In some aspects, L is selected from

[0106] wherein:

[0107] q is an integer from 2 to 10;

[0108] — is absent or a bond;

[0109] the point of attachment to X; and*

[0110] is the point of attachment to the binding moiety.[OHl] In some aspects, L is

[0113] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0114] A is phenyl;

[0115] U is NH;

[0116] R1is halo; and

[0117] X is -N(R200)v(CH2)m”O(CH2)n”-; wherein:

[0118] v is 1;

[0119] m’ ’ and n’ ’ are 2; and

[0120] R200is methyl.

[0121] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0122] A is phenyl;

[0123] U is NH;

[0124] R1is halo; and

[0125] X is -N(R200)v(CH2)m”O(CH2)n”-; wherein:

[0126] v is 2;

[0127] m’ ’ and n’ ’ are 2; and

[0128] each R200is methyl.

[0129] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0130] A is phenyl;

[0131] U is NH;

[0132] R1is halo; and

[0133] X is -O(CH2)n”-; wherein:

[0134] n” is 2.

[0135] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0136] A is phenyl;

[0137] U is NH;

[0138] R1is halo; and

[0139] X is -S(CH2)n”-; wherein:

[0140] n” is 2.

[0141] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0142] A is phenyl;

[0143] U is NH;

[0144] R1is hydrogen; and

[0145] X is -NR200-; wherein:

[0146] R200is methyl.

[0147] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0148] A is phenyl;

[0149] U is NH;

[0150] R1is halo; and

[0151] X is -NR200-; wherein:

[0152] R200is hydrogen.

[0153] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0154] A is phenyl;

[0155] U is NH;

[0156] R1is halo; and

[0157] X is -NR2-; wherein:

[0158] R2is hydrogen.

[0159] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0160] A is phenyl;

[0161] U is NH;

[0162] R1is hydrogen; and

[0163] X is -C(CH3)=

[0164] In some aspects, the conjugate is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0165] A is a C4-Ciocycloalkyl ring;

[0166] U is NH;

[0167] R1is hydrogen; and

[0168] X is -N(R200)(CH2)mO(CH2)n-; wherein:

[0169] n” is 1;

[0170] m” is 2; and

[0171] R200is methyl.

[0172] In some aspects, the conjugate is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is cleavable by a protease.

[0173] In some aspects, L50is selected from the group consisting of

[0175] wherein:

[0176] q is from 2 to 10;

[0177] Z1, Z2, Z3, Z4, and Z5are each independently absent or a naturally-occurring amino acid residue in the L- or D-configuration, provided that at least two of Z1, Z2, Z3, Z4, and Z5are amino acid residues;

[0178] is the point of attachment to the parent molecular moiety; and

[0179] is the point of attachment to the binding moiety.

[0180] In some aspects, Z1, Z2, Z3, Z4, and Z5are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D- glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D- asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; provided that at least two of Z1, Z2, Z3, Z4, and Z5are amino acid residues.

[0181] In some aspects:

[0182] Z1is absent or glycine;

[0183] Z2is absent or selected from the group consisting of L-glutamine, D-glutamine, L- glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine;

[0184] Z3is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine;

[0185] Z4is selected from the group consisting of L-citrulline, D-citrulline, L-asparagine,D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine; and

[0186] Z5is absent or glycine.

[0187] In some aspects, L50is

[0189] In some aspects, q is 4.

[0190] In some aspects, the conjugate is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein L50is a bioreducible linker.

[0191] In some aspects, L50is selected from the group consisting of

[0194] q is from 2 to 10;

[0195] R, R’, R”, and R’” are each independently selected from hydrogen, Ci-CealkoxyCi-Cealkyl, (Ci-Cealkyl^NCi-Cealkyl, and Ci-Cealkyl, or, two geminal R groups, together with the carbon atom to which they are attached, can form a cyclobutyl or cyclopropyl ring;

[0196] is the point of attachment to the parent molecular moiety; and

[0197] is the point of attachment to the binding moiety.

[0198] In some aspects, L50is

[0199]

[0200] In some aspects, q is 2.

[0201] In some aspects, the conjugate is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein L50is a click-to-release linker.

[0202] In some aspects, L50is

[0203]

[0204] wherein:

[0205] q is from 2 to 10;

[0206] is the point of attachment to the parent molecular moiety; and

[0207] is the point of attachment to the binding moiety.

[0208] In some aspects, the conjugate is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein L50is a beta-glucuronidase cleavable linker.

[0209] In some aspects, L50is selected from

[0212] wherein:

[0213] q is from 2 to 10;

[0214] — is absent or a bond;

[0215] is the point of attachment to the parent molecular moiety; and

[0216] is the point of attachment to the binding moiety.

[0217] In some aspects, a is from 3 to 4.

[0218] In some aspects, a is 8.

[0219] In some aspects, L or L50is attached to a cysteine in the binding moiety, optionally wherein the conjugation is irreversible.

[0220] In some aspects, the present disclosure provides a conjugate which is:

[0222] wherein Bm is antibody CD 123.001 and a is 2-8, optionally 3-5

[0223] In some aspects, the present disclosure provides a composition comprising at least one conjugate described herein, wherein the average number of neodegraders per Bm is about 2 to about 8, optionally wherein the average number of neodegraders per Bm is about 3 to about 5 or wherein the average number of neodegraders per Bm is about 3.5 to 4.5.

[0224] In some aspects, the present disclosure provides a method of treating a hematological malignancy in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of an antibody or antigen-binding fragment thereof, conjugate or a pharmaceutically acceptable salt thereof, or a composition described herein.

[0225] In some aspects, the hematological malignancy is CD 123 -positive hematological malignancy.

[0226] In some aspects, the hematological malignancy is a leukemia or a lymphoma.

[0227] In some aspects, the hematological malignancy is an acute myeloid leukemia (AML), B- cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, Hodgkin lymphoma, or blastic plasmacytoid dendritic neoplasm (BPDCN).

[0228] In some aspects, the hematological malignancy is present as minimal residual disease (MRD).

[0229] In some aspects, the hematological malignancy is AML. In some aspects, the AML is disseminated AML.

[0230] In some aspects, the subject is an unfit subject.

[0231] In some aspects, the hematological malignancy has a TP53 mutation.

[0232] In some aspects, the hematological malignancy has a FLT3 mutation, optionally wherein the in the mutation is an internal tandem duplication (ITD).

[0233] In some aspects, the subject has previously been treated with venetoclax and hypomethylating agent (HMA).

[0234] In some aspects, the present disclosure provides the use of an antibody or antigen-binding fragment thereof, a conjugate or a pharmaceutically acceptable salt thereof, or a composition described herein in the preparation of a medicament for use any of the methods described herein.

[0235] In some aspects, the present disclosure provides an antibody or antigen-binding fragment thereof, a conjugate or a pharmaceutically acceptable salt thereof, or a composition for use in any of the methods described herein.

[0236] In some aspects, the present disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding fragment thereof described herein.

[0237] In some aspects, the present disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region or light chain of the antibody or antigen-binding fragment thereof described herein.

[0238] In some aspects, the present disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof described herein.

[0239] In some aspects, the present disclosure provides an isolated vector comprising a polynucleotide described herein.

[0240] In some aspects, the present disclosure provides a host cell comprising (a) a polynucleotide described herein, (b) a vector described herein, or (c) a first vector comprising a polynucleotide described herein and a second vector comprising a polynucleotide described herein.

[0241] In some aspects, the host cell is a CHO cell or a CHO-K1 cell.

[0242] In some aspects, the present disclosure provides a method of producing an antibody or antigen-binding fragment thereof that immunospecifically binds to CD 123 comprising culturing a host cell described herein so that the nucleic acid molecule is expressed and the antibody or antigen-binding fragment thereof is produced, optionally wherein the method further comprises isolating the antibody or antigen-binding fragment thereof from the culture.

[0243] In some aspects, the present disclosure provides an antibody or antigen-binding fragment thereof that immunospecifically binds to CD123 and is encoded by a polynucleotide described herein or produced by a method described herein.

[0244] In some aspects, the present disclosure provides a method of making a conjugate comprising conjugating an antibody or antigen-binding fragment thereof described herein to a GSPT1 degrader.

[0245] In some aspects, the present disclosure provides a conjugate produced by a method described herein.BRIEF DESCRIPTION OF THE FIGURES

[0246] FIG. 1A shows binding of anti-CD123 antibodies to recombinant human CD 123.

[0247] FIG. IB shows binding of anti-CD123 antibodies to recombinant mouse and cynomolgus CD123.

[0248] FIG. 2 shows cell surface binding of anti-CD123 antibodies to MV4-11 AML cells.

[0249] FIG. 3 shows binding of the 32703 antibody and a conjugate thereof with Compound (I) (i.e., Conjugate A) to recombinant human CD123.

[0250] FIG. 4A shows the in vitro potency of the 32703 antibody and a conjugate thereof with Compound (I) (i.e., Conjugate A) against MV4-11 AML cells.

[0251] FIG. 4B shows the in vitro potency of the 32703 antibody and a conjugate thereof with Compound (I) (i.e., Conjugate A) against TOM-1 B-ALL cells.

[0252] FIG. 5 shows the in vivo efficacy of conjugates containing Compound (I) and either the 32703 antibody (Conjugate A) or the 18G3 antibody (see U.S. Patent No. 11,613,581 at SEQ ID NOs: 27 and 30), which contains an IgGl LLQG sequence (Conjugate B), in an MV4-11 luciferase cell line xenograft model.

[0253] FIG. 6 shows the body weight analysis in the MV4-11 luciferase cell line xenograft model.

[0254] FIG. 7 shows the N-glycosylation of LC in 32703 variants as analyzed by SDS-PAGE analysis.

[0255] FIG. 8 shows binding of 32703 variants to recombinant human CD123 as measured by ELISA.

[0256] FIG. 9 shows the internationalization rates of anti-CD123 antibodies h7G3, 32703, and 32703 variants.

[0257] FIG. 10A shows binding of anti-CD123 antibodies to cynomolgus CD 123.

[0258] FIG. 10B shows binding of anti-CD123 antibodies to mouse CD 123.

[0259] FIG. 11 shows cell surface binding of anti-CD123 antibodies to MV4-11 AML cells.

[0260] FIG. 12 shows binding of anti-CD123 antibody CD123.001 and a conjugate thereof with Compound (I) (i.e., Conjugate C) to recombinant human CD123.

[0261] FIG. 13 shows competitive binding between anti-CD 123 antibodies.

[0262] FIG. 14 shows the in vitro potency of anti-CD 123 GSPT1 neodegrader conjugates against MV4-11 AML cells.

[0263] FIG. 15 shows GSPT1 degradation after exposure to anti-CD 123 antibody CD 123.001 and a conjugate thereof with Compound (I) (i.e., Conjugate C) in MV4-11 AML cells as measured by Western blot analysis. Conjugate C showed higher GSPT1 degradation activity than the GSPT1 Neodegrader (XI) or CC-885 at the matched dose.

[0264] FIG. 16 shows the effect of anti-CD123 antibodies on MV4-11 cell viability in the presence and absence of IL-3.

[0265] FIG. 17 shows the effect of the anti-CD123 GSPT1 neodegrader Conjugate C on MV4- 11 cell viability in the presence and absence of IL-3.

[0266] FIG. 18A is a graph showing the in vivo efficacy of anti-CD 123 GSPT1 neodegrader conjugates in an MV4-11 luciferase cell line xenograft model.

[0267] FIG. 18B depicts visual images showing the in vivo efficacy of anti-CD123 GSPT1 neodegrader conjugates in an MV4-11 luciferase cell line xenograft model.

[0268] FIG. 19 shows the Kaplan-Meier survival curve following administration of anti-CD123 GSPT1 neodegrader conjugates in the MV4-11 luciferase cell line xenograft model.

[0269] FIG. 20 shows the body weight analysis in the MV4-11 luciferase cell line xenograft model.

[0270] FIG. 21 provides a comparison of the internalization efficiency of several CD123 antibodies.

[0271] FIG. 22 shows the binding of CD 123 conjugates to CD 123.

[0272] FIG. 23 shows the in vitro cytotoxicity of Conjugate C as compared to other test articles in AML cell lines.

[0273] FIG. 24 shows the in vitro cytotoxicity of Conjugate C in HL and B-ALL cell lines.

[0274] FIG. 25 shows the in vitro cytotoxicity of Conjugate C in a TP53 isogenic model.

[0275] FIG. 26 shows the in vitro cytotoxicity of various CD 123 conjugates.

[0276] FIGs. 27A and 27B show the effects of Conjugate C on human normal myeloid and erythroid progenitors.

[0277] FIG. 28A is a graph showing the minimal efficacious dose of Conjugate C compared to the standard of care in an MV4-11 luciferase cell line xenograft model.

[0278] FIG. 28B depicts visual images showing the AML cancer burden of Conjugate C in an MV4-11 luciferase cell line xenograft model.

[0279] FIG. 29 shows the body weights throughout the treatment cycle in the MV4-11 luciferase cell line xenograft modelDETAILED DESCRIPTION

[0280] The present disclosure provides binding moieties that are capable of specifically binding to CD123. In some aspects, the binding moiety is an antibody or an antigen-binding fragment.

[0281] The present disclosure also provides to a conjugate of formula (I) or formula (II):(II), or a pharmaceutically acceptable salt thereof, wherein:

[0282] a is from 1 to 10;

[0283] n is O or l;

[0284] A is phenyl or a C4-Ciocycloalkyl ring;

[0285] U is selected from NH, O, S, and CF2;

[0286] R1is independently selected from hydrogen and halo;

[0287] R10is selected from -CH3, -C(O)R30, -N(R40)2, -(CH2)n OH, -(CH2)n’N(R40)2,-(CH2)n Q(CH2)m OH, -(CH2)n Q(CH2)m SH, and -(CH2)n Q(CH2)mN(R40)2; wherein:

[0288] R30is hydrogen or Ci-Cealkyl;

[0289] each R40is independently hydrogen or Ci-Cealkyl;

[0290] Q is O, S, or NR40;

[0291] n’ is 1-6; and

[0292] m’ is 2-5;

[0293] R20is selected from hydrogen, -(CH2CH2O)V-CH3, C2-Cealkenyl, Ci-Cealkyl; C2-Cealkynyl, benzyl, C3-Cecycloalkyl, and C3-C6cycloalkyl(Ci-C3alkyl), wherein v’ is from 1 to 24;

[0294] R50and R51are independently selected from hydrogen and deuterium;

[0295] X is selected from -NR200-, =C(CH3)-, -Q’-(CH2)n”-, and -Q’(CH2)m”Q”(CH2)n”-; wherein:

[0296] Q’ and Q” are each independently O, S, or N(R200)v, wherein:

[0297] v is 1 or 2; and

[0298] each R200is independently hydrogen or Ci-Cealkyl;

[0299] n” is an integer from 1 to 6; and

[0300] m’ ’ is an integer from 2 to 6;

[0301] wherein the left side of each group is attached to L and the right side is attached to A;

[0302] provided that when X is NH or -Q’-(CH2)n ”-, R1is halo;

[0303] each Y is independently S or O;

[0304] L is a cleavable linker or non-cleavable linker;

[0305] L50is a cleavable linker;

[0306] and

[0307] Bm is a binding moiety that is capable of specifically binding to CD 123. In some aspects, the binding moiety is an antibody or an antigen-binding fragment.

[0308] The present disclosure also provides compositions comprising the conjugates, and the method of using and making the conjugates.I. Definitions

[0309] In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0310] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences.As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a negative limitation.

[0311] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0312] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0313] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0314] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0315] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0316] The terms “targeted protein degrader,” and “neoDegrader,” as used herein, refer to a molecule that forms a ternary complex with an E3 ubiquitin ligase which is capable of targeting a protein for degradation. Examples include, but are not limited to, molecular glues and PROTACs. Examples of molecular glues include, but are not limited to CC-90009, lenalidomide, pomalidomide, mezigdomide (CC-92480), iberdomide (CC-220), DKY709, and Compound Pl disclosed in WO2021 / 198965.

[0317] The term “DAR,” as used herein, refers to the drug antibody ratio of conjugates, which is the average number of neoDegrader-linker complexes linked to each binding moiety (e.g., antibody or antigen-binding fragment thereof). In certain aspects, the DAR of the conjugates described herein is from 1 to 10. In some aspects, the DAR of the conjugates described herein is from 1 to 8. In some aspects, the DAR of the conjugates described herein is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.

[0318] The term “antibody,” as used herein, also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cells. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.

[0319] An “intact antibody” is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.

[0320] “Antibody fragments” comprise a portion of an intact antibody, generally the antigenbinding or variable region thereof. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0321] The term “single domain antibody,” also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of from about 12kDa to about 15kDa. Single body antibodies can be based on heavy chain variable domains or light chains. Examples of single domain antibodies include, but are not limited to, VHH fragments and VNAR fragments.

[0322] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method, or may be made by recombinant DNA methods. The “monoclonal antibodies” may also be isolated from phage antibody libraries.

[0323] The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

[0324] Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans. Another method to prepare MAbs uses genetic engineering including recombinant DNA techniques. Monoclonal antibodies made from these techniques include, among others, chimeric antibodies and humanized antibodies. A chimeric antibody combines DNA encoding regions from more than one type of species. For example, a chimeric antibody may derive the variable region from a mouse and the constant region from a human. A humanized antibody comes predominantly from a human, even though it contains nonhuman portions. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. But unlike a chimeric antibody, the variable region may be partially derived from a human. The nonhuman, synthetic portions of a humanized antibody often come from CDRs in murine antibodies. In any event, these regions are crucial to allow the antibody to recognize and bind to a specific antigen. While useful for diagnostics and short-term therapies, murine antibodies cannot be administered to people long-term without increasing the risk of a deleterious immunogenic response. This response, called Human Anti-Mouse Antibody (HAMA), occurs when a human immune system recognizes the murine antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death.

[0325] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the nonhuman portions of administered antibodies. Furthermore, chimeric and humanized antibodies can have the additional benefit of activating secondary human immune responses, such as antibody dependent cellular cytotoxicity.

[0326] The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0327] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes”. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0328] An antibody or antigen-binding fragment thereof that “immunospecifically binds” a molecular target or an antigen of interest (e.g., CD123) is one capable of binding to that target or antigen with sufficient affinity and specificity so that the binding to that target or antigen is measurably different from a non-specific interaction and such that the antibody or fragment is useful in targeting a cell expressing the antigen.

[0329] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In certain aspects, the term “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Reference to “about” a value or parameter herein also includes (and describes) reference to that value or parameter per se.

[0330] The terms “administration,” “administering,” and grammatical variants thereof refer to introducing a composition, such as an antibody, antigen-binding fragment thereof, or conjugate of the present disclosure, into a subject via a pharmaceutically acceptable route. The introduction into a subject is by any suitable route, including parenterally (e.g., intravenously). Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function.

[0331] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0332] As used herein, the term “conserved” refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.

[0333] In some aspects, two or more sequences are said to be “completely conserved” or “identical” if they are 100% identical to one another. In some aspects, two or more sequences are said to be “highly conserved” if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to one another. In some aspects, two or more sequences are said to be “conserved” if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to one another. Conservation of sequence can apply to the entire length of a polynucleotide or polypeptide or can apply to a portion, region or feature thereof.

[0334] The term “GSPT1 neoDegrader conjugate” as used herein refers to a GSPT1 neoDegrader attached to a binding moiety (e.g., an antibody or antigen-binding fragment thereof) through a linker.

[0335] As used herein, the terms “linking” and “conjugating” are used interchangeably an each refer to the covalent or non-covalent attachment of two or more moi eties comprising a neoDegrader and a binding moiety (e.g., an antibody or antigen-binding fragment thereof). In some aspects the

[0336] The term “amino acid sequence variant” refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% sequence identity with at least one receptorbinding domain of a native antibody or with at least one ligand binding domain of a native receptor, and typically, they will be at least about 80%, more typically, at least about 90% homologous by sequence with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by the conventional names, one-letter and three-letter codes.

[0337] “Sequence identity” is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for the alignment are well known in the art. One such computer program is “Align 2,” authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

[0338] The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. Moreover, a FcR may be one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITEM) in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.

[0339] “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed.

[0340] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0341] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P- sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0342] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0343] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whosename reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0344] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0345] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0346] The “light chains” of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0347] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen-binding.

[0348] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0349] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanization is a method to transfer the murine antigen-binding information to a non-immunogenic human antibodyacceptor, and has resulted in many therapeutically useful drugs. The method of humanization generally begins by transferring all six murine complementarity determining regions (CDRs) onto a human antibody framework. These CDR-grafted antibodies generally do not retain their original affinity for antigen-binding, and in fact, affinity is often severely impaired. Besides the CDRs, select non-human antibody framework residues must also be incorporated to maintain proper CDR conformation. The transfer of key mouse framework residues to the human acceptor in order to support the structural conformation of the grafted CDRs has been shown to restore antigen-binding and affinity. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0350] An “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain aspects, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, or more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0351] A “cancer” refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. A “hematological cancer” or “blood cancer” refers to a cancer that begins in a blood-forming tissue such as the bone marrow or in the cells of the immune system.

[0352] A “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs. In some aspects, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

[0353] The term “therapeutically effective amount” or “therapeutically effective dosage” refers to an amount of an agent (e.g., binding molecule or GSPT1 neoDegrader conjugate disclosed herein) that provides the desired biological and / or therapeutic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay cancer recurrence. An effective amount can be administered in one or more administrations. The effective amount of the composition can, for example, (i) reduce the number of cancer cells; and / or (ii) relieve to some extent one or more of the symptoms associated with the cancer.

[0354] The terms “treat” or “treatment” refer to measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.IL Anti-CD123 Binding Moieties

[0355] The term “binding moiety,” as used herein, refers to any molecule that recognizes and binds to a cell surface marker or receptor. The binding moiety can have certain therapeutic effect such as anti-proliferative (cytostatic and / or cytotoxic) activity against a target cell or pathway. In certain aspects the binding moiety can comprise or can be engineered to comprise at least one chemically reactive group such as a carboxylic acid, amine, thiol, or chemically reactive amino acid moiety or side chain. Following specific binding or complexing of a binding moiety orconjugate provided herein with CD123, a cell expressing CD123 is permissive for uptake of the neoDegrader conjugate, which is then internalized into the cell.

[0356] In some aspects, group “Bm” can be a moiety that can specifically bind to CD 123. In some aspects, group “Bm” can be a peptide or a protein that binds to CD 123. In certain aspects, group “Bm” can be an antibody, antibody fragment, or an antigen-binding fragment, e.g., an anti- CD123 antibody or antigen-binding fragment thereof as disclosed herein.

[0357] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to CD 123. Antibodies and antigen-binding fragments thereof can be murine, human, humanized, chimeric, or derived from other species.

[0358] Monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g., CD123). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV-hybridoma technique. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this disclosure may be cultivated in vitro or in vivo.

[0359] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art.

[0360] In some aspects, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion may be with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. The first heavy-chain constant region (CHI) may contain a site necessary for light chain binding. Nucleic acids with sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, can be inserted into one or more expression vectors and co-transfected into a suitable host cell.

[0361] Useful antibody fragments include functionally active fragments, derivatives or analogs of an antibody that immunospecifically bind to CD123. For example, useful antibodies fragments include fragments such as, but not limited to, F(ab’)2 fragments, which contain the variable region, the light chain constant region and the CHI domain of the heavy chain, can be produced by pepsindigestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab’)2 fragments. Other useful antibody fragments are heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SC As), or any other molecule with the same specificity as the antibody.

[0362] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0363] Completely human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the disclosure. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0364] Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. Human antibodies can also be produced using various techniques known in the art, including phage display libraries.

[0365] The antibody can be a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portionthereof, such as at least 10, 20 or 50 amino acid portion of the protein) that is not the antibody. The antibody or fragment thereof may be covalently linked to the other protein at the N-terminus of the constant domain.

[0366] Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen-binding immunospecificity. For example, but not by way of limitation, the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.

[0367] The antibodies can include antibodies having modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor. Antibodies immunospecific for CD123 can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.

[0368] In certain aspects, the antibody can be a monoclonal antibody, e.g., a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. In some aspects, the antibody can be an antibody fragment, e.g., a Fab fragment.

[0369] Antibodies immunospecific for a CD 123 -expressing cancer cell can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for CD 123 can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.

[0370] In some aspects, an antibody or antigen-binding fragment thereof that binds to CD 123 comprises the 6 CDRs of an antibody in Table A (i.e., the 3 CDRs of the variable heavy chain or heavy chain and the 3 CDRs of the variable light chain or light chain of the same antibody).

[0371] The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In some aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises the 6 Kabat-defined CDRs of an antibody in Table A (i.e., the 3 Kabat-defined CDRs of the variable heavy chain or heavy chain and the 3 Kabat-defined CDRs of the variable light chain or light chain of the same antibody).

[0372] The CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273 : 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontane K etal., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0373] In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises the 6 Chothia-defined CDRs of an antibody in Table A (i.e., the 3 Chothia-defined CDRs of the variable heavy chain or heavy chain and the 3 Chothia-defined CDRs of the variable light chain or light chain of the same antibody). In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence. In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises a combination of Kabat CDRs and Chothia CDRs of an antibody in Table A.

[0374] In some aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et aL, (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL- CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises the 6 IMGT-defined CDRs of an antibody in Table A (i.e., the 3 IMGT-defined CDRs of the variable heavy chain or heavy chain and the 3 IMGT-defined CDRs of the variable light chain or light chain of the same antibody), for example, as described in Lefranc M-P (1999) supra and Lefranc M-P et aL, (1999) supra).

[0375] In some aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises the 6 MacCallum-defmed CDRs of an antibody in Table A (i.e., the 3 MacCallum- defined CDRs of the variable heavy chain or heavy chain and the 3 MacCallum-defmed CDRs of the variable light chain or light chain of the same antibody), for example as determined by the method in MacCallum RM et al.

[0376] In some aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In someaspects, a binding moiety is an antibody or antigen-binding fragment thereof that comprises the 6 AbM-defined CDRs of an antibody in Table A (i.e., the 3 AbM-defined CDRs of the variable heavy chain or heavy chain and the 3 AbM-defined CDRs of the variable light chain or light chain of the same antibody) as determined by the AbM numbering scheme.

[0377] In some aspects, a binding moiety is an antibody or antigen-binding fragment thereof that binds to CD 123. In some aspects, an antibody or antigen-binding fragment thereof binds to CD 123 and comprises the 6 CDRs of an anti-CD123 antibody provided in Tables A and B (i.e., the 3 VH CDRs of an anti-CD123 antibody in Table A and the 3 VL CDRs of the same antibody in Table B; e.g., 6 CDRs comprising the amino acid sequences of SEQ ID NOs:2-7).

[0378] In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH of an anti-CD123 antibody provided in Table C (e.g., a VH comprising the amino acid sequence of SEQ ID NO:21). In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:21. In some aspects, an antibody or antigenbinding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:21. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:21. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:21. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:21. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:21.

[0379] In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VL of an anti-CD123 antibody provided in Table C (e.g., a VL comprising the amino acid sequence of SEQ ID NO:22). In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigenbinding fragment thereof binds to CD123 and comprises a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibodyor antigen-binding fragment thereof binds to CD 123 and comprises a VL comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VL comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VL comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:22.

[0380] In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH and a VL of an anti-CD123 antibody provided in Table C (i.e., the VH of an anti- CD123 antibody in Table C and a VL of the same antibody in Table C; e.g., a VH comprising the amino acid sequence of SEQ ID NO:21 and a VL comprising the amino acid sequence of SEQ ID NO:22). In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:21 and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:21 and a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:21 and a VL comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:21 and a VL comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:21 and a VL comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a VH comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:21 and aVL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:22.

[0381] In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a heavy chain comprising the amino acid sequences of SEQ ID NOs:21 and 51 and a light chain comprising the amino acid sequence of SEQ ID NO:22.

[0382] In some aspects, an antibody or antigen-binding fragment thereof binds to CD123 and comprises a heavy chain comprising the amino acid sequences of SEQ ID NO:55 and a light chain comprising the amino acid sequence of SEQ ID NO:56.Table A: Exemplary Anti-CD 123 Heavy CDR SequencesTable B: Exemplary Anti-CD 123 Light CDR SequencesTable C: Exemplary Anti-CD 123 Variable SequencesTable D: Exemplary Anti-CD 123 Full Length Sequences

[0383] In some aspects, an antibody or antigen-binding fragment thereof comprises a constant region. In some aspects, an antibody or antigen-binding fragment thereof comprises a CHI domain. In some aspects, an antibody or antigen-binding fragment thereof comprises a CH2 domain. In some aspects, an antibody or antigen-binding fragment thereof comprises a CH3 domain. In some aspects, an antibody or antigen-binding fragment thereof comprises a CL domain.

[0384] In some aspects, an antibody or antigen-binding fragment thereof comprises a CHI domain, CH2 domain, and a CH3 domain. In some aspects, an antibody or antigen-binding fragment thereof comprises a CHI domain, CH2 domain, a CH3 domain, and a CL domain.

[0385] In some aspects, a constant region, a CHI domain, a CH2 domain, a CH3 domain, or a CL domain is an engineered constant region, CHI domain, CH2 domain, CH3 domain or a CL domain.

[0386] In some aspects, an antibody or antigen-binding fragment thereof comprises a heavy chain constant region, e.g., a human heavy chain constant region. In some aspects, an antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region, e.g., a human IgGheavy chain constant region. In some aspects, an antibody or antigen-binding fragment thereof comprises an IgGl heavy chain constant region, e.g., a human IgGl heavy chain constant region. In some aspects, an antibody or antigen-binding fragment thereof comprises an IgG4 heavy chain constant region.

[0387] In some aspects, an antibody or antigen-binding fragment thereof comprises a light chain constant region, e.g., a human light chain constant region. In some aspects, an antibody or antigenbinding fragment thereof comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In some aspects, an antibody or antigen-binding fragment thereof comprises a gamma light chain constant region, e.g., a human gamma light chain constant region.

[0388] In some aspects, an antibody or antigen-binding fragment thereof comprises an engineered cysteine at heavy chain position S239 according to EU numbering. In some aspects, an antibody or antigen-binding fragment thereof comprises an engineered cysteine at heavy chain position K334 according to EU numbering.

[0389] In some aspects, an antibody or antigen-binding fragment there comprises an Fc domain with reduced effector function as compared to a wild-type Fc domain. Such antibodies or antigenbinding fragments thereof can have reduced toxicity. In some aspects, an antibody or antigenbinding fragment thereof comprises an N297A mutation according to EU numbering. In some aspects, an antibody or antigen-binding fragment thereof comprises a LALA (L234A and L235A) mutation according to EU numbering. In some aspects, an antibody or antigen-binding fragment thereof comprises an N297A mutation according to EU numbering and a LALA (L234A and L235A) mutation according to EU numbering.

[0390] Accordingly, an antibody or antigen-binding fragment thereof can comprise a heavy chain constant region of SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49.IgGl Heavy Chain Constant RegionASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG (SEQ ID NO:47)IgGl Heavy Chain Constant Region S239CASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG (SEQ ID NO:48)IgGl Heavy Chain Constant Region K334CASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI< ALPAPIECTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:49)

[0391] An antibody or antigen-binding fragment thereof can comprise a heavy chain constant region of SEQ ID NO:50.IgG4 Heavy Chain Constant Region S228PASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NQ:50)

[0392] An antibody or antigen-binding fragment thereof can comprise a heavy chain constant region of SEQ ID NO:51.IgGl N297A Constant regions (CH1-Hinge-CH2-CH3)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:51)

[0393] An antibody or antigen-binding fragment thereof can comprise a heavy chain constant region of SEQ ID NO:52.IgGl L234A, L235A Constant regions (CH1-Hinge-CH2-CH3)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG (SEQ ID NO:52)

[0394] An antibody or antigen-binding fragment thereof can comprise a heavy chain constant region of SEQ ID NO:53.IgGl L234A, L235A, N297A Constant regions (CH1-Hinge-CH2-CH3)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG (SEQ ID NO:53)

[0395] An antibody or antigen-binding fragment thereof can comprise a light chain constant region of SEQ ID NO:54.RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO:54)III. GSPT1 NeoDegrader Conjugates

[0396] The present disclosure provides conjugates of one or more GSPT1 neoDegraders and an anti-CD123 binding moiety disclosed herein. These conjugates can degrade proteins by binding to cereblon (CRBN), promoting recruitment and ubiquitination of substrate proteins mediated by CRL4CRBNE3 ubiquitin ligase. These agents act as “molecular glues,” filling the binding interface as a hydrophobic patch that reprograms protein interactions between the ligase and neosubstrates.

[0397] The binding moiety, in addition to potentially having certain therapeutic effect such as anti-proliferative (cytostatic and / or cytotoxic) activity against a target cell or pathway, can also target the neoDegrader to a specific cell, tissue, or location.

[0398] In some aspects, the present disclosure provides a conjugate of formula (I) or formula (II),or a pharmaceutically acceptable salt thereof, wherein:

[0399] a is from 1 to 10;

[0400] n is O or l;

[0401] A is phenyl or a C4-Ciocycloalkyl ring;

[0402] U is selected from NH, O, S, and CF2;

[0403] R1is independently selected from hydrogen and halo;

[0404] R10is selected from -CH3, -C(O)R30, -N(R40)2, -(CH2)n OH, -(CH2)n’N(R40)2, -(CH2)n Q(CH2)m OH, -(CH2)n Q(CH2)m SH, and -(CH2)n’Q(CH2)mN(R40)2, wherein:

[0405] R30is hydrogen or Ci-Cealkyl;

[0406] each R40is independently hydrogen or Ci-Cealkyl;

[0407] Q is O, S, or NR40;

[0408] n’ is 1-6; and

[0409] m’ is 2-5;

[0410] R20is selected from hydrogen, -(CH2CH2O)v -CH3, C2-Cealkenyl, Ci-Cealkyl; C2- Cealkynyl, benzyl, Cs-Cecycloalkyl, and C3-C6cycloalkyl(Ci-C3alkyl), wherein v’ is from 1 to 24;

[0411] R50and R51are independently selected from hydrogen and deuterium;

[0412] X is selected from -NR200-, =C(CH3)-, -Q’-(CH2)n”-, and -Q’(CH2)m”Q”(CH2)n”-; wherein:

[0413] Q’ and Q” are each independently O, S, or N(R200)v; wherein:

[0414] v is 1 or 2; and

[0415] each R200is independently hydrogen or Ci-Cealkyl;

[0416] n” is an integer from 1 to 6; and

[0417] m’ ’ is an integer from 2 to 6;

[0418] wherein the left side of each X group is attached to L and the right side is attached to A;

[0419] provided that when X is NH or -Q’-(CH2)n ”-, R1is halo;

[0420] each Y is independently S or O;

[0421] L is a cleavable linker or non-cleavable linker;

[0422] L50is a cleavable linker;

[0423] and

[0424] Bm is a binding moiety that is capable of specifically binding to CD123, e.g., as described above.

[0425] In some aspects, R50and R51are each hydrogen.

[0426] In some aspects, R50and R51are each deuterium.

[0427] In some aspects, one of R50and R51is deuterium and the other is hydrogen.III.A. GSPT1 NeoDegraders

[0428] A GSPT1 neoDegrader degrades “G1 to S phase transition protein 1” homolog (GSPT1). Human GSPT1 has been assigned UniProt accession number P15170. Human GSPT1 can have the amino acid sequence of SEQ ID NO:1.MELSEPIVENGETEMSPEESWEHKEEISEAEPGGGSLGDGRPPEESAHEMMEEEEEIPKPKSVVAPPGAPKKEHVNVVFIGHVDAGKSTIGGQIMYLTGMVDKRT LEKYEREAKEKNRETWYLSWALDTNQEERDKGKTVEVGRAYFETEKKHFTI LDAPGHKSFVPNMIGGASQADLAVLVISARKGEFETGFEKGGQTREHAMLAK TAGVKHLIVLINKMDDPTVNWSNERYEECKEKLVPFLKKVGFNPKKDIHFMP CSGLTGANLKEQSDFCPWYIGLPFIPYLDNLPNFNRSVDGPIRLPIVDKYKDMGTVVLGKLESGSICKGQQLVMMPNKHNVEVLGILSDDVETDTVAPGENLKIR LKGIEEEEILPGFILCDPNNLCHSGRTFDAQIVIIEHKSIICPGYNAVLHIHTCIEE VEIT ALICE VDKKSGEKSKTRPRFVKQDQVCIARLRTAGTICLETFKDFPQMG RFTLRDEGKTIAIGKVLKLVPEKD (SEQ ID NO:1)

[0429] In some aspects, the present disclosure provides GSPT1 neoDegraders of formula (X):and / or pharmaceutically acceptable salts thereof; wherein:

[0430] n is O or l;

[0431] A is phenyl or a C4-Ciocycloalkyl ring;

[0432] U is selected from NH and CF2;

[0433] R1is independently selected from hydrogen and halo;

[0434] R10is selected from -CH3, -C(O)R30, -N(R40)2, -(CH2)n OH, -(CH2)nN(R40)2, -(CH2)n Q(CH2)m OH, -(CH2)n Q(CH2)m SH, and -(CH2)n’Q(CH2)mN(R40)2; wherein:

[0435] R30is hydrogen or Ci-Cealkyl;

[0436] each R40is independently hydrogen or Ci-Cealkyl;

[0437] Q is O, S, or NR40;

[0438] n’ is 1-6; and

[0439] m’ is 2-5;

[0440] R50and R51are each independently hydrogen or deuterium; and

[0441] each Y is independently S or O.

[0442] In some aspects, R50and R51are each hydrogen.

[0443] In some aspects, R50and R51are each deuterium.

[0444] In some aspects, one of R50and R51is deuterium and the other is hydrogen.

[0445] As used herein, the term “C2-C6alkenyl” refers to a group derived from a straight or branched chain hydrocarbon containing from two to six carbon atoms and at least one carboncarbon double bond.

[0446] As used herein, the term “Ci-Cealkoxy,” as used herein, refers to a Ci-Cealkyl group attached through an oxygen atom.

[0447] As used herein, the term “Ci-CealkoxyCi-Cealkyl” refers to a Ci-Cealkoxy group attached through a Ci-Cealkyl group.

[0448] As used herein, the term “Ci-Cealkynyl” refers to a group derived from a straight or branched chain hydrocarbon containing from two to six carbon atoms and at least one carboncarbon triple bond.

[0449] As used herein, the term “C2-C6alkenyl” refers to a group derived from a straight or branched chain saturated hydrocarbon containing from two to six carbon atoms and at least one carbon-carbon double bond.

[0450] As used herein, the term “Cs-Cecycloalkyl” refers to a saturated monocyclic, hydrocarbon ring system having three to six carbon atoms and zero heteroatoms. Representative examples include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl.

[0451] As used herein, the term “C4-Ciocycloalkyl” refers to a saturated monocyclic, hydrocarbon ring system having four to ten carbon atoms and zero heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl groups containing between seven and ten atoms may be monocyclic or fused, spirocyclic, or bridged bicyclic structures.

[0452] As used herein, the term “C3-C6cycloalkyl(Ci-C3alkyl)” refers to a C3-Cecycloalkyl group attached through a Ci-C3alkyl group.

[0453] As used herein, the term “halo” refers to F, Cl, Br, or I.

[0454] In some aspects, the present disclosure provides GSPT1 neoDegraders of formula (XI):(XI).III.B. Linkers

[0455] The neoDegraders of the present disclosure can be linked to the binding moiety via a linker. As used herein, the term “linker” refers to any chemical moiety capable of connecting the binding moiety (Bm) to group X within the conjugates of formula (I) or to the nitrogen atom of the glutaramide ring within the conjugates of formula (II).

[0456] In certain aspects, the linkers can contain a heterobifunctional group. In the present disclosure, the term “heterobifunctional group” refers to a chemical moiety that connects the linker of which it is a part to the binding moiety. Heterobifunctional groups are characterized as having different reactive groups at either end of the chemical moiety. Attachment to “Bm,” can be accomplished through chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the binding moiety with a reaction handle on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine coupling, and coupling via a non-natural amino acid incorporated by genetic engineering, wherein non-natural amino acid residues with a desired reaction handle are installed onto “Bm.” In enzymatic conjugation, an enzyme mediates the coupling of the linker with an accessible amino residue on the binding moiety. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation may also be used sequentially. For example, enzymatic conjugation can also be used for installing unique reaction handles on “Bm” to be utilized in subsequent chemical conjugation.

[0457] In some aspects, the heterobifunctional group is selected from:wherein:

[0458] is the point of attachment to the remaining portion of the linker; and

[0459] * the point of attachment to Bm.

[0460] In certain aspects, linker “L” is non-cleavable. As used here, the term “non-cleavable linker” is any chemical moiety that is capable of linking the binding moiety to the neoDegrader in a stable, covalent manner and does not fall under the categories defined herein as “cleavable linkers”. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light- induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. “Substantially resistant to cleavage” means that the chemical bond in the linker or adjoining the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the antibody neoDegrader conjugate population remains non-cleavable by an acid, a photolabile-cleaving agent, a bioreductive agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves the chemical bond (for example, a disulfide bond) in a cleavable linker, for within a few hours to several days of treatment with any of the agents described above. In certain aspects the linker is not susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the neoDegrader and / or binding moiety can remain active. NeoDegrader conjugate catabolites generated from non-cleavable linkerscontain a residual amino acid from the antibody. These catabolites can exert unique and unexpected properties in the target cells to which they are delivered.

[0461] A person of ordinary skill in the art would readily distinguish non-cleavable from cleavable linkers.

[0462] Examples of non-cleavable linkers include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate) linkers, succinimide thioether linkers, and linkers such as:

[0464] the point of attachment to X; and

[0465] is the point of attachment to the binding moiety.

[0466] In some aspects, L is:

[0467] In some aspects, p is 5.

[0468] In certain aspects L and L50can be cleavable linkers. In some aspects, the linkers can be susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the neoDegrader and / or binding moiety can remain active.

[0469] In some aspects, the cleavable linkers can be cleaved enzymatically. In some aspects, the cleavable linker can be cleaved by a protease, peptidase, esterase, beta-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase.

[0470] In some aspects, the cleavable linkers can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like.

[0471] In certain aspects, the cleavable linkers contain a peptide. In some aspects, the peptide is the site of cleavage of the linker, thereby facilitating release of the drug upon exposure to intracellular proteases, such as lysosomal enzymes. Peptides can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. Examples of peptides having two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalaninehomolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides having three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine-alanine-asparagine (ala-ala-asn), alanine- phenylalanine-lysine (ala-phe-lys), glycine-glycine-phenylalanine (gly-gly-phe), and glycine- glycine-glycine (gly-gly-gly). Examples of peptides having four amino acids include, but are not limited to, glycine-glycine-valine-citrulline (gly-gly-val-cit) and glycine-glycine-phenylalanine- glycine (gly-gly-phe-gly). Examples of peptides having five amino acids include, but are not limited to, glycine-glycine-valine-citrulline-glycine (gly-gly-val-cit-gly) and glycine-glycine- phenylalanine-glycine-glycine (gly-gly-phe-gly-gly). The amino acid combinations above can also be present in the reverse order (i.e., cit-val).

[0472] The peptides of the present disclosure can comprise L- or D- isomers of amino acid residues. The term “naturally-occurring amino acid” refers to Ala, Asp, Asx, Cit, Cys, Glu, Phe, Glx, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, and Tyr. “D-” designates an amino acid having the “D” (dextrorotary) configuration, as opposed to the configuration in the naturally occurring (“L-”) amino acids. The amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0473] In certain aspects, linkers L and L50are protease cleavable linkers selected fromwherein:

[0474] q is an integer from 2 to 10;

[0475] Z1, Z2, Z3, Z4, and Z5are each independently absent or a naturally-occurring amino acid residue in the L- or D-configuration, provided that at least two of Z1, Z2, Z3, Z4, and Z5are amino acid residues;

[0476] is the point of attachment to group X within the conjugates of formula (I) or to the nitrogen atom of the glutaramide ring within the conjugates of formula (II); and

[0477] is the point of attachment to the binding moiety.

[0478] In certain aspects, Z1, Z2, Z3, Z4, and Z5are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L- glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L- asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; provided that at least two of Z1, Z2, Z3, Z4, and Z5are amino acid residues.

[0479] In some aspects, Z1is absent or glycine; Z2is absent or selected from L-glutamine, D- glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D- alanine, and glycine; Z3is selected from L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z4is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine; and Z5is absent or glycine.

[0480] In some aspects, L is

[0481] In some aspects, q is 5.

[0482] In some aspects, L50is

[0483] In some aspects, q is 4.

[0484] In certain aspects, L is a pyrophosphatase cleavable linker.

[0485] In some aspects, L is a pyrophosphatase cleavable linker which is:wherein:

[0486] q is an integer from 2 to 10;

[0487] the point of attachment to X; and

[0488] is the point of attachment to the binding moiety.

[0489] In certain aspects, L is a beta-glucuronidase cleavable linker.

[0490] In some aspects, L is a beta-glucuronidase cleavable linker selected from:wherein:

[0491] q is an integer from 2 to 10;

[0492] — is absent or a bond;

[0493] the point of attachment to X; and *

[0494] is the point of attachment to the binding moiety.

[0495] In some aspects, L is

[0496] In certain aspects, L50is a beta-glucuronidase cleavable linker.

[0497] In some aspects, L50is a beta-glucuronidase cleavable linker selected from

[0498] q is from 2 to 10;

[0499] — is absent or a bond;

[0500] is the point of attachment to the nitrogen atom of the glutaramide ring within the conjugates of formula (II); and

[0501] is the point of attachment to the binding moiety.

[0502] In some aspects, L and L50are bioreducible linkers. Bioreducible linkers take advantage of the difference in reduction potential in the intracellular compartment versus plasma. Reduced glutathione presented in tumor cells’ cytoplasm is up to 1000-fold higher than that present in normal cells’ cytoplasm, and the tumor cells also contain enzymes which can contribute to reduction in cellular compartments. The linkers keep conjugates intact during systemic circulation,and are selectively cleaved by the high intracellular concentration of glutathione, releasing the active drugs at the tumor sites from the non-toxic prodrugs.

[0503] In some aspects, L and L50are bioreducible linkers selected from:wherein:

[0504] q is an integer from 2 to 10;

[0505] R, R’, R”, and R’” are each independently selected from hydrogen, Ci-CealkoxyCi-Cealkyl, (Ci-Cealkyl^NCi-Cealkyl, and Ci-Cealkyl, or, two geminal R groups, together with the carbon atom to which they are attached, can form a cyclobutyl or cyclopropyl ring;

[0506] is the point of attachment to group X within the conjugates of formula (I) or to the nitrogen atom of the glutaramide ring within the conjugates of formula (II); and

[0507] is the point of attachment to the binding moiety.

[0508] In some aspects, L50is

[0509] In some aspects, q is 2.

[0510] In certain aspects, L is an acid-cleavable linker. Acid-cleavable linkers are specifically designed to remain stable at the neutral pH of blood circulation, but undergo hydrolysis and release the cytotoxic drug in the acidic environment of the cellular compartments.

[0511] In some aspects, L is an acid-cleavable linker selected fromwherein:

[0512] q is an integer from 2 to 10;

[0513] the point of attachment to X; and *

[0514] is the point of attachment to the binding moiety.

[0515] In certain aspects, L and L50are click-to-release linkers, where release of the neoDegrader is chemically triggered by a tetrazine or related compound.

[0516] In some aspects, Land L50are click-to-release linkers selected fromwherein:

[0517] q is an integer from 2 to 10;

[0518] is the point of attachment to group X within the conjugates of formula (I) or to the nitrogen atom of the glutaramide ring within the conjugates of formula (II); and

[0519] is the point of attachment to the binding moiety.III. C. Linker Atachment to Binding Moiety

[0520] A linker can be attached to an amino acid in the binding moiety, e.g., an amino acid in the constant region of binding moiety that is an antibody or antigen-binding fragment thereof. For example, a linker can be attached to an amino acid in a CHI domain. A linker can be attached to an amino acid in a CH2 domain. A linker can be attached to an amino acid in a CH3 domain. A linker can be attached to an amino acid in a CL domain.

[0521] In some aspects, a linker can be attached to an amino acid in a heavy chain constant region, e.g., a human heavy chain constant region. In some aspects, a linker can be attached to an amino acid in an IgG heavy chain constant region, e.g., a human IgG heavy chain constant region. In some aspects, a linker can be attached to an amino acid in an IgGl heavy chain constant region, e.g., a human IgGl heavy chain constant region. In some aspects, a linker can be attached to an amino acid in an IgG4 heavy chain constant region, e.g., a human IgG4 heavy chain constant region.

[0522] In some aspects, a linker can be attached to an amino acid in a light chain constant region, e.g., a human light chain constant region. In some aspects, a linker can be attached to an amino acid in a kappa light chain constant region, e.g., a human kappa light chain constant region. In some aspects, a linker can be attached to an amino acid in a gamma light chain constant region, e.g., a human gamma light chain constant region.

[0523] In some aspects, the linker is irreversibly conjugated to the anti-CD123 antibody or antigen-binding fragment thereof. Methods for irreversible conjugation are disclosed, for example, in Chen F-J and Gao J, Chemistry. 2022 November 25; 28(66): e202201843. doi: 10.1002 / chem.202201843, which is herein incorporated by reference in its entirety.

[0524] In some aspects, a linker is attached to a cysteine in an anti-CD123 antibody or antigenbinding fragment thereof. In some aspects, the cysteine is in a constant region of the anti-CD123 antibody or antigen-binding fragment thereof.

[0525] In some aspects, a linker can be attached to an engineered cysteine in an anti-CD123 antibody or antigen-binding fragment thereof, e.g., at heavy chain position S239C according to EU numbering. In some aspects, a linker can be attached to an engineered cysteine at heavy chain position K334C according to EU numbering in an anti-CD123 antibody or antigen-binding fragment thereof.

[0526] In some aspects, a linker can be attached to heavy chain Q295 of an anti-CD123 antibody or antigen-binding fragment thereof according to EU numbering.III.D. GSPT1 Neodegraders Per Conjugate

[0527] In the present disclosure, group “Bm” can be conjugated to more than one neoDegrader. In some aspects, “Bm” can be conjugated to from 1 to 10 neoDegraders. In some aspects, “Bm” can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 neoDegraders. In some aspects, “Bm” can be conjugated to from 1 to 9 neoDegraders. In some aspects, “Bm” can be conjugated to from 1 to 8 neoDegraders. In some aspects, “Bm” can be conjugated to from 2 to 9 neoDegraders. In some aspects, “Bm” can be conjugated to from 2 to 8 neoDegraders. In some aspects, “Bm” is conjugated to 3 neoDegraders. In some aspects, “Bm” is conjugated to 4 neoDegraders. In some aspects, “Bm” is conjugated to 5 neoDegraders. In some aspects, “Bm” is conjugated to 6 neoDegraders. In some aspects, “Bm” is conjugated to 7 neoDegraders. In some aspects, “Bm” is conjugated to 8 neoDegraders. In some aspects, “Bm” is conjugated to 9 neoDegraders.IV Compositions and Methods of Using

[0528] The binding moieties and conjugates described herein can be in the form of pharmaceutically acceptable salts. In some aspects, such salts are derived from inorganic or organic acids or bases.

[0529] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy ethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl -propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

[0530] Examples of suitable base addition salts include ammonium salts; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; salts with organic bases, such as dicyclohexylamine salts, A-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, and the like.

[0531] For example, Berge lists the following FDA-approved commercially marketed salts: anions acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedi sulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate,glutamate, glycollylarsanilate (glycollamidophenylarsonate), hexylresorcinate, hydrabamine (MAf’-di (dehydroabietyl)ethylenedi amine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate (methanesulfonate), methylbromide, methylnitrate, methyl sulfate, mucate, napsylate (2- naphthalenesulfonate), nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate (8- chlorotheophyllinate) and triethiodide; organic cations benzathine (N,N’~ dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine ( / f-methylglucamine) and procaine; and metallic cations aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

[0532] Berge additionally lists the following non-FDA-approved commercially marketed (outside the United States) salts: anions adipate, alginate, aminosalicylate, anhydromethylenecitrate, arecoline, aspartate, bisulfate, butylbromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), napadisylate (1,5 -naphthalenedi sulfonate), oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate and undecanoate; organic cations benethamine (A-benzylphenethylamine), clemizole (l-p-chlorobenzyl-2-pyrrolildine-l’- ylmethylbenzimidazole), diethylamine, piperazine and tromethamine (tris(hydroxymethyl)aminomethane); and metallic cations barium and bismuth.

[0533] Pharmaceutical compositions comprising the neoDegrader conjugates described herein may also comprise suitable carriers, excipients, and auxiliaries that may differ depending on the mode of administration.

[0534] In some aspects, the pharmaceutical compositions can be formulated as a suitable parenteral dosage form. Said formulations can be prepared by various methods known in the art. The pharmaceutical compositions can be administered parentally. Suitable means for parenteral administration include intravenous administration. Suitable devices for parenteral administration include needle injectors, needle-free injectors, and infusion techniques.

[0535] Parenteral compositions are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents. However, the composition may also be formulated a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile pyrogen-free water.

[0536] The preparation of parenteral compositions under sterile conditions, for example, by lyophilization, can be readily accomplished using standard techniques known well to those of skill in the art.

[0537] The parenteral formulations can be admixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms such as, but not limited to, preservatives.

[0538] Compositions comprising anti-CD123 neoDegrader conjugates described herein can have a drug antibody ration (DAR) of about 2 to about 8. Such compositions can also have a DAR of about 2 to about 5. Such compositions can also have a DAR of about 3 to about 8. Such compositions can also have a DAR of about 3 to about 5. Such compositions can also have a DAR of about 3.5 to about 4.5.

[0539] The neoDegrader conjugates of the present disclosure can be used as medicaments such as agents for the treatment of diseases, for example, hematological cancers — e.g., CD 123 -positive cancers. In some aspects, the cancer is a hematological malignancy (e.g., a leukemia or a lymphoma). The hematological malignancy can be, for example, an acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, Hodgkin lymphoma, or blastic plasmacytoid dendritic neoplasm (BPDCN).

[0540] In some aspects, a subject that can be treated with a conjugate of the present disclosure is a nonhuman animal such as a rat or a mouse. In some aspects, the subject that can be treated is a human.V. Methods of Preparing NeoDegraders and Compositions

[0541] The compounds of the present disclosure can be prepared by one of ordinary skill in the art in light of the present disclosure and knowledge in the art, and / or by reference to the schemes shown below and the synthetic examples. Exemplary synthetic routes are set forth in schemes below and in Examples. It should be understood that the variables, (for example “R” groups) appearing in the following schemes and examples are to be read independently from those appearing elsewhere in the application. One of ordinary skill in the art would readily understand how the schemes and examples shown below illustrate the preparation of the compounds described herein.EXAMPLESExample 1 : Synthesis of Compound (I)

[0542] Compound (I) was prepared using the procedure described in WO2022 / 254376, which is incorporated herein by reference in its entirety.Example 1-1 : Synthesis of Compound (1-1)

[0543] Compound (1-1) was prepared using the procedure described in WO2021 / 198965, which is incorporated herein by reference in its entirety.Example 1-2: Synthesis of Compound (1-2)Compound (1-2) was prepared using the procedure described in WO2021 / 198965, which is incorporated herein by reference in its entirety.Example 1-3: Synthesis of Compound (1-3)

[0544] Compound (1-3) was prepared using the procedure described in WO2021 / 198965, which is incorporated herein by reference in its entirety.Example 1-4: Synthesis of Compound (1-4)

[0545] Step 1 : To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (6.00 g, 27.83 mmol, 1.00 equiv) in THF (120.00 mL) were added BH3-Me2S(34.7 mL, 2 M in Me2S, 69.4 mmol, 2.50 equiv) dropwise. The resulting mixture was stirred for 2h at 70°C. The mixture was cool down to room temperature. TLC (PE: EtOAc = 3: 1) indicated the reaction was completed. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with PEZEtOAc (1 : 1) to afford 2-(2-chloro-4- nitrophenyl)ethanol (3.99 g, 71%) as a yellow solid.XH NMR (300 MHz, DMSO) 5 8.24 (s, 1H), 8.14-8.11 (m, 1H), 7.67-7.64(m, lH),4.87(t, J =12Hz, 1H), 3.70-3.64 (m,2H), 2.99-2.94(m ,2H).

[0546] Step 2: To a stirred solution of PPhs (1.95 g, 7.44 mmol, 1.50 equiv) in DCM (20.00 mL) were added NBS (1.32 g, 7.44 mmol, 1.50 equiv) dropwise at room temperature. The resulting mixture was stirred for 30min at room temperature. 2-(2-chloro-4-nitrophenyl)ethanol (1.00 g, 4.96 mmol, 1.00 equiv) added in portions to the stirred mixture. The resulting mixture was stirred for 3h at room temperature. TLC (PE: EtOAc = 10: 1) indicated the reaction was completed. The mixture was added water (30.00 mL). The aqueous layer was extracted with CH2CI2 (3x30 mL). The combined organic layer was washed with water, brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with PEZEtOAc (10: 1) to afford l-(2-bromoethyl)-2-chloro-4- nitrobenzene (1.13 g, 86%) as an off-white solid.1HNMR (300 MHz, DMSO) 5 8.29 (s, 1H), 8.14- 8.11 (m, 1H), 7.52-7.49(m, 1H), 3.69-3.64 (m, 2H), 3.44-3.40(m , 2H).

[0547] Step 3: To a stirred solution of methyl thioglycolate (1.37 g, 12.9 mmol, 3.00 equiv) in MeOH (25.00 mL) were added NaOMe(0.46 g, 8.54 mmol, 2.00 equiv) dropwise at room temperature. The resulting mixture was stirred for Ih at 0 °C. l-(2-bromoethyl)-2-chloro-4- nitrobenzene(l .13 g, 4.27 mmol, 1.00 equiv) were added to the above stirred mixture. The resulting mixture was stirred for 3h at room temperature. TLC ((PE: EtOAc = 10: 1) indicated the reaction was completed. The mixture was added water (20.00 mL). The aqueous layer was extracted with EtOAc (3x30 mL). The combined organic layer was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with PEZEtOAc (5: 1) to afford methyl 2-[[2- (2-chloro-4-nitrophenyl)ethyl]sulfanyl]acetate(1.0g, 81%) as a yellow solid. ’H NMR (400 MHz, CDCh) 5 8.22(d, J=2.4Hz, IH), 8.06(dd, J=8.0, 2.4Hz, IH), 7.46(d, J=8.0Hz, IH), 4.22(s, 3H), 3.24(s, 2H), 2.94(d, J=8.0Hz, 2H), 2.90(d, J=8.0Hz, 2H).

[0548] Step 4: To a stirred solution of methyl 2-[[2-(2-chloro-4- nitrophenyl)ethyl]sulfanyl]acetate(3.00 g, 10.35 mmol, 1.00 equiv) in THF (60.00 mL) were addedLiOH (0.50 g, 19.25 mmol, 1.85 equiv) in JLO (10.00 mL) dropwise at 0 °C. The resulting mixture was stirred for 2h at Odegrees C. LCMS indicated the reaction was completed. The mixture was added water (40.00 mL), adjusted pH to 5 with HC1 (IN), extracted with CH2CI2 (3x80 mL). The combined organic layer was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with CH2CI2 / MeOH (20: 1) to afford [[2-(2-chloro-4- nitrophenyl)ethyl]sulfanyl]acetic acid (2.7 g, 95%) as a yellow solid. LCMS (ES, m / z): 274[M- H] .

[0549] Step 5: To a stirred solution of [[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]acetic acid (2.80 g, 10.15 mmol, 1.00 equiv) in DMF (60.00 mL) were added methanamine, hydrochloride (0.82 g, 12.18 mmol, 1.20 equiv) and HATU (5.79 g, 15.23 mmol, 1.50 equiv) and DIEA (5.25 g, 40.62 mmol, 4.00 equiv) at 0 °C . The resulting mixture was stirred for overnight at room temperature. LCMS indicated the reaction was completed. The mixture was added water(50.00 mL). The aqueous layer was extracted with EtOAc (3x100 mL). The combined organic layer was washed with water, brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under vacuum. The residue was purified by reverse flash chromatography with the following conditions: C18 column; mobile phase, ACN in water (0.1%FA), 0% to 50% gradient in 40 min; detector, UV 254 nm. This resulted in 2-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-N- methylacetamide (2.8 g, 95%) as a yellow solid. LCMS (ES, m / z): 289,291[M+H]+.

[0550] Step 6: To a stirred solution of 2-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-N- methylacetamide(320.00 mg, 1.10 mmol, 1.00 equiv) in THF (8.00 mL) were added BH3-THF (1.37 mL, 2 M in THF, 2.74mmol, 2.50 equiv) dropwise at room temperature. The resulting mixture was stirred for 4h at 70 °C. The mixture was cool down to room temperature. LCMS indicated the reaction was completed. MeOH (4.00 mL) and 1 N HC1 were added to the mixture at 70 °C. The mixture was cooled down to room temperature. The mixture was added water (20.00 mL). The aqueous layer was extracted with CH2CI2 (3x20 mL). The combined organic layer was washed with water, brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under vacuum. This resulted in (2-[[2-(2-chloro-4- nitrophenyl)ethyl]sulfanyl]ethyl)(methyl)amine (290 mg, 95%) as a yellow solid. LCMS (ES, m / z): 275,277[M+H]+.

[0551] Step 7: To a stirred solution of (2-[[2-(2-chloro-4- nitrophenyl)ethyl]sulfanyl]ethyl)(methyl)amine(300.00 mg, 1.09 mmol, 1.00 equiv) and NaHC03(353.00 mg, 3.27 mmol, 3.00 equiv) in water were added BOC2O (238.00 mg, 1.09 mmol,1.00 equiv) in THF (4.00 mL) dropwise at room temperature. The resulting mixture was stirred for 4 h at room temperature. LCMS indicated the reaction was completed. The aqueous layer was extracted with CH2CI2 (3x20 mL). The combined organic layer was washed with water, brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (8: 1) to afford tert-butyl N-(2-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethyl)-N-methylcarbamate (220 mg, 53%) as a yellow solid. LCMS (ES, m / z): 375,377[M+H]+, 275,277[M+H-100]+

[0552] Step 8: To a stirred solution of tert-butyl N-(2-[[2-(2-chloro-4- nitrophenyl)ethyl]sulfanyl]ethyl)-N-methylcarbamate (374.00 mg, 0.99 mmol, 1.00 equiv) in EtOH (3.75 mL) were added NH4Q (103.00 mg, 1.92 mmol, 1.93 equiv) in H2O (0.75 mL) and Fe (180.00 mg, 3.22 mmol, 3.23 equiv) dropwise at room temperature. The resulting mixture was stirred for 3h at 70 °C under nitrogen atmosphere. LCMS indicated the reaction was completed. The mixture was cool down to room temperature. The reaction mixture was filtered. The filtrate was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (10: 1) to afford tert-butyl N-(2-[[2-(4-amino-2- chlorophenyl)ethyl]sulfanyl]ethyl)-N-methylcarbamate (140 mg, 40%) as a yellow oil. LCMS (ES, m / z): 345,347[M+H]+, 245,247[+H-100]+.

[0553] Step 9: To a stirred mixture of tert-butyl N-(2-[[2-(4-amino-2- chlorophenyl)ethyl]sulfanyl]ethyl)-N-methylcarbamate (140.00 mg, 0.40 mmol, 1.00 equiv) in THF (3.00 mL) were added diphosgene (50.00 uL, 0.40 mmol, 1.00 equiv) dropwise at 0 °C. The resulting mixture was stirred for Ih at Odegree C. The resulting mixture was concentrated under vacuum and re-dissolved in DMF (2 mL). To a stirred mixture of 3-[5-(aminomethyl)-l-oxo-3H- isoindol-2-yl]piperidine-2, 6-dione (112.00 mg, 0.40 mmol, 1.00 equiv) and TEA (80.00 mg, 0.80 mmol, 2 equiv) in DMF (3.00 mL) were added the solution mentioned above dropwise at 0 degrees C. The resulting mixture was stirred for 3h at degrees C. LCMS indicated the reaction was completed. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water(0.1%FA), 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in tert-butyl N-[2-[(2-[2-chloro-4-[([[2-(2,6- dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5- yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]ethyl]-N-methylcarbamate (160 mg, 61%) as a yellow oil. LCMS (ES, m / z): 644,646[M+H]+, 544,546[M+H-100]+.

[0554] Step 10: To a stirred solution of tert-butyl N-[2-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin- 3-yl)-l-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]ethyl]-N- methyl carbamate (175.00 mg, 1 equiv) in DCM (1.50 mL) were added TFA (0.50 mL) dropwise at 0 °C. The resulting mixture was stirred for 3 h at 0 °C. LCMS indicated the reaction was completed. The resulting mixture was concentrated under vacuum. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 min; detector, UV 254 nm. This resulted in 1- [3-chloro-4-(2-[[2-(methylamino)ethyl]sulfanyl]ethyl)phenyl]-3-[[2-(2,6-dioxopiperidin-3-yl)-l- oxo-3H-isoindol-5-yl]methyl]urea (65 mg, 44%) as a yellow oil. LCMS (ES, m / z): 544,546[M+H]+.

[0555] Step 11 : To a stirred solution of l-[3-chloro-4-(2-[[2-(methylamino)ethyl]sulfanyl]ethyl)phenyl]-3-[[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5- yl]methyl]urea (65.00 mg, 0.12 mmol, 1.00 equiv) and [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6- (2,5-dioxopyrrol-l-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl 4- nitrophenyl carbonate(106.00 mg, 0.14 mmol, 1.20 equiv) in DMF (1.00 mL) were added HOBT (97.00 mg, 0.72 mmol, 6.00 equiv) and 2,6-lutidine (77.00 mg, 0.72 mmol, 6.00 equiv) dropwise at 0 °C . The resulting mixture was stirred for overnight at room temperature. LCMS indicated the reaction was completed. The reaction mixture was purified by Prep-HPLC with the following conditions (Column: Xselect CSH OBD Column 30x150mm 5um, Mobile Phase A:Water(0.1%FA), Mobile Phase B:ACN; Flow rate:60 mL / min; Gradient:28 B to 52 B in 7 min; 220 nm; RTl :6.88min; The collected fraction was lyophilized to afford [4-[(2S)-5- (carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-l-yl)hexanamido]-3- methylbutanamido]pentanamido]phenyl]methyl N-[2-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3- yl)-l-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]ethyl]-N- methylcarbamate (20.9 mg, 15%) as a white solid. LCMS (ES, m / z): 1142,1144[M+H]+,XH NMR (300 MHz, DMSO) 511.01 (s, lH)10.01 (s, 1H), 9.90(s, 1H), 8.80 (s, 1H), 8.20-8.00(m, 1H), 7.81(d, J =8.4 Hz, 1H), 7.70-7.43 (m, 6H), 7.28-7.15 (m, 4H), 7.00 (s, 2H), 6.83-6.81 (m, 1H), 5.98-6.96(m, 1H), 5.41(s, 2H), 5.41-5.01 (m, 1H), 4.99 (s, 2H), 4.47-7.19 (m, 6H), 3.39-3.30 (m, 4H), 3.10-2.87 (m, 8H), 2.76-2.62 (m, 5H), 2.41-2.25 (m, 1H), 2.25-2.09 (m, 2H), 2.08-1.97 (m, 2H), 1.97-1.51 (m, 2H), 1.51-1.44 (m, 6H), 1.20-1.18 (m, 2H), 0.86-0.65 (m, 6H).Example 1-5: Synthesis of Compound (1-5)

[0556] Compound (1-5) was prepared using the procedure described in WO2021 / 198965, which is incorporated herein by reference in its entirety.Example 1-6: Synthesis of Compound (1-6)

[0557] Compound (1-6) was prepared using the procedure described in WO2021 / 198965, which is incorporated herein by reference in its entirety.Example 1-7: Synthesis of Compound (1-7)

[0558] Step 1 : To a stirred mixture of NaNs (8.00 g, 123.0 mmol, 1.25 equiv) in H2O (80.0 mL) was added ethyl (2E)-4-bromobut-2-enoate (20.00 g, 98.4 mmol, 1.00 equiv) in acetone (120.0 mL) at 0 °C. The resulting mixture was stirred for 3 h at 60 °C. The mixture was allowed to cool down to room temperature. The resulting mixture was extracted with EtOEt (3 x 60 mL). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in ethyl (2E)-4- azidobut-2-enoate (15 g, 78%) as a yellow oil. LCMS (ES, m / z): 156 [M+H]+

[0559] Step 2: To a stirred mixture of ethyl (2E)-4-azidobut-2-enoate (15 g, 87.0 mmol, 1 equiv) in THF (190 mL) was added ethanethioic O-acid (10.6 g, 139.2 mol, 1.60 equiv) and DIEA (15.7 g, 121.8 mmol, 1.40 equiv) dropwise at 0 °C. The resulting mixture was stirred for 1 h at 0 °C. The resulting mixture was stirred for 16 h at 25 °C. The reaction was quenched by the addition of water (100 mL) at room temperature. The resulting mixture was extracted with CH2CI2 (3 x 200 mL). The combined organic layers were washed with NaEICCh, NaEEPCh, brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PEZEtOAc (6:1) to afford ethyl 3- (acetylsulfanyl)-4-azidobutanoate (6 g, 26 %) as a yellow oil. LCMS (ES, m / z): 232 [M+H]+

[0560] Step 3: To a stirred mixture of ethyl 3-(acetylsulfanyl)-4-azidobutanoate (6.00 g, 20.7 mmol, 1.00 equiv) in H2O2 (35.0 mL) was added HO Ac (104.0 mL) dropwise at 25 °C. The resulting mixture was stirred for 16 h at 25 °C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2CI2 / MeOH (5: 1) to afford l-azido-4-ethoxy-4-oxobutane-2-sulfonic acid (5 g, 71%) as a yellow oil. LCMS (ES, m / z): 236 [M-H]+

[0561] Step 4: A mixture of l-azido-4-ethoxy-4-oxobutane-2-sulfonic acid (4.00 g, 15.1 mmol, 1.00 equiv) in HC1 (3 M) (60.0 mL) and dioxane (60.00 mL) was stirred for 6 h at 80 °C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. This resulted in 4-azido-3 -sulfobutanoic acid (3 g, 66%) as a yellow oil. LCMS (ES, m / z): 208 [M-H]'

[0562] Step 5: To a solution of 4-azido-3 -sulfobutanoic acid (3.00 g, 12.9 mmol, 1.00 equiv) in MeOH (150.0 mL) was added Pd / C (10%) (0.30 g, 2.84 mmol, 0.22 equiv) under nitrogen atmosphere. The mixture was hydrogenated at room temperature for 16 h under hydrogen atmosphere using a hydrogen balloon, filtered through a diatomaceous earth pad and concentratedunder reduced pressure. This resulted in 4-amino-3 -sulfobutanoic acid (2.8 g, 82%) as a white solid. LCMS (ES, m / z): 182 [M-H]'

[0563] Step 6: To a stirred mixture of 4-amino-3 -sulfobutanoic acid (2.50 g, 12.2 mmol, 1.00 equiv) in H2O (40 mL) was added NaHCOs (2.06 g, 24.5 mmol, 2.00 equiv) in portions at 0 °C. To the above mixture was added methyl 2,5-dioxopyrrole-l-carboxylate (3.81 g, 0.025 mmol, 2.00 equiv) at 0 °C. The resulting mixture was stirred for additional 4 h at 25 °C. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water (containing 0.1% FA), ACN (5% to 10% gradient in 20 min; detector, UV 220 nm. This resulted in 4-(2,5-dioxopyrrol-l-yl)-3-sulfobutanoic acid (2 g, 43%) as a yellow oil. LCMS (ES, m / z): 262 [M-H]’

[0564] Step 7: To a stirred mixture of 4-(2,5-dioxopyrrol-l-yl)-3-sulfobutanoic acid (2.00 g, 6.83 mmol, 1.00 equiv) in DMF (40 mL) was added DCC (2.20 g, 10.6 mmol, 1.56 equiv) at 0 °C. The resulting mixture was stirred for 10 min at 0 °C. To the above mixture was added pentafluorophenol (1.60 g, 0.009 mmol, 1.27 equiv) at 0 °C. The resulting mixture was stirred for additional 4 h at 25 °C. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water (containing 0.1% FA), ACN (5% to 10% gradient in 20 min; detector, UV 220 nm. This resulted in l-(2,5-dioxopyrrol-l-yl)-4-oxo-4-(2,3,4,5,6- pentafluorophenoxy)butane-2-sulfonic acid (300 mg, 7%) as a yellow oil. LCMS (ES, m / z): 428 [M-H]'

[0565] Step 8: To a stirred mixture of l-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)- 3-[[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5-yl]methyl]urea hydrochloride (100.00 mg, 0.15 mmol, 1.00 equiv) and DIEA (124.00 mg, 0.95 mmol, 6.0 equiv) in DMF (1.70 mL) was added l-(2,5-dioxopyrrol-l-yl)-4-oxo-4-(2,3,4,5,6-pentafluorophenoxy)butane-2-sulfonic acid (137.00 mg, 0.31 mmol, 2.00 equiv) at 25 °C. The resulting mixture was stirred for 3 h at 25 °C. The crude product was purified by Prep-HPLC with the following conditions: Column, YMC- Actus Triart C18, 20 x 250 mm, 5 um, 12 nm; mobile phase, water (containing 0.1% FA) and ACN (20% up to 40% in 7 min); Detector, UV 254 nm. The collected fraction was lyophilized to afford l-[[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5- yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]-3-(2,5-dioxopyrrol-l- yl)propane-2-sulfonic acid (37.3 mg, 30%) as a white solid. LCMS (ES, m / z): 773 [M+H]+; ’H- NMR (DMSO, 400 MHz) 5 (ppm): 10.98 (s, 1H), 8.79 (d, J= 7.2 Hz, 1H), 7.70-7.67 (m, 2H), 7.51 (s, 1H), 7.44 (d, J= 8.0 Hz, 1H), 7.23-7.18 (m, 1H), 7.14-7.08 (m, 1H), 6.88-6.83 (m, 3H), 5.11-, 4H), 3.83-3.79 (m, 1H), 3.55-3.45 (m, 4H), 3.33-3.26 (m, 3H), 3.20-, 5H), 2.52-2.50 (m, 3H), 2.49-2.37 (m, 1H), 2.33-2.31 (m, 1H), 2.01-Example 1-8: Synthesis of Compound (1-8)

[0566] Step 1 : To solution of (4-bromo-2-chlorophenyl)acetic acid(25.00 g, 100.20 mmol, 1.00 equiv) in THF(500 mL) was added solution of BH3-Me2S(19 mL, 200.44 mmol, 2.00 equiv) at 0 °C. The reaction was put into a 70 °C oil bath and stirred at this temperature for 3 h. TLC (PE:EA=4: 1) indicated the reaction was completed. The reaction was quenched with methanol and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =4: 1) to give 2-(4-bromo-2-chlorophenyl)ethanol (21 g, 80%) as a yellow oil. ’H NMR (300 MHz, DMSO-de) 5 7.68 (d, J=2.1 Hz, 1H), 7.49 (dd, J=8.2, 2.1 Hz, 1H), 7.33 (d, J=8.2 Hz, 1H), 4.74 (t, J=5.3 Hz, 1H), 3.61 (td, J=6.9, 5.3 Hz, 2H), 2.3 (t, J=6.9 Hz, 2H).

[0567] Step 2: To a solution of 2-(4-bromo-2-chlorophenyl)ethanol (21.00 g, 88.98 mmol, 1.00 equiv) in toluene (200 mL) was added tert-butyl 2-bromoacetate(139.14 g, 713 mmol, 8.00 equiv), BU4NHSO4 (24.22 g, 713 mmol, 0.80 equiv) and a solution of NaOH (17.83 g, 446 mmol, 5.00 equiv) in water(200 mL) at room temperature in air. The resulting mixture was stirred at room temperature for 2 h. TLC(PE: EtOAc =5: 1) indicated the reaction was completed. The organic phase was separated out and the water phase was extracted with EA (50 mL).The combined organic layer was washed with water (100 mL), brine (100 mL), dried over anhydrous sodium sulfate and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =5: 1) to give tert-butyl 2-[2-(4-bromo-2- chlorophenyl)ethoxy]acetate (10 g, 30% ) as a colorless oil.1HNMR (300 MHz, Chloroform-d) 5 7.53 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.2, 2.0 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 3.98 (s, 2H), 3.75 (t, J = 6.3 Hz, 2H), 3.04 (t, J = 6.9 Hz, 2H), 1.45 (s, 9H).

[0568] Step 3: 2-[2-(4-bromo-2-chlorophenyl)ethoxy]acetate (10 g) in DCM (100 ml) was TFA (10 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 h. LCMS indicated the reaction was completed. The resulting mixture was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =1 :2) to give [2-(4-bromo-2-chlorophenyl)ethoxy]acetic acid (3.8 g, 45% ) as yellow oil. LCMS (ES, m / z):291[M-H]'

[0569] Step 4: To a solution of [2-(4-bromo-2-chlorophenyl)ethoxy]acetic acid (3.79 g, 12.91 mmol, 1.00 equiv) in DMF (40 mL) were added HATU (7.36 g, 19.37 mmol, 1.5 equiv), methanamine, hydrochloride (1.05 g, 15.49 mmol, 1.20 equiv) and DIEA (5.01 g, 38.73 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred at room temperature for 3 h. LCMS indicated the reaction was completed. The reaction was diluted with water (400 mL),the resulting mixture was extracted with EA (200 mL*3), the combined organic layer was wahsed with water (200 ml), brine (200 mL), dried over anhydrous sodium sulfate and concentrated to dryness. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =1 :2) to give 2-[2-(4-bromo-2-chlorophenyl)ethoxy]-N-methylacetamide (3.5 g, 87% ) as a yellow oil. LCMS (ES, m / z):306[M+H]+

[0570] Step 5: To a sealed tube a solution of 2-[2-(4-bromo-2-chlorophenyl)ethoxy]-N- methylacetamide (1.16 g, 3.78 mmol, 1.00 equiv)*3 in THF (20 mL) *3 and BEE-THF (19 mL, 18.9 mmol, 5.00 equiv)*3 was added at room temperature under N2.Then the reaction was stirred at 700C for 16 h. LCMS indicated the reaction was completed. After cooled to room temperature. The reaction was quenched by the addition of methanol. The resulting mixture was concentrated to dryness under vacuum, the residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water(0.1%FA), 0% to 60% gradient in 30 min; detector, UV 254 nm to give {2-[2-(4-bromo-2- chlorophenyl)ethoxy]ethyl}(methyl)amine ( 2.01 g,60%) as a yellow oil. LCMS (ES, m, / z):292[M+H]+.

[0571] Step 6: To a solution of {2-[2-(4-bromo-2-chlorophenyl)ethoxy]ethyl}(methyl)amine (2 g, 6.84 mmol, 1.00 equiv) in THF (20 mL) were added BOC2O (1.64 g, 7.51 mmol, 1.10 equiv) and solutio of NaHCOs (0.86 g, 10.24 mmol, 1.50 equiv) in water (20 mL) at room temperature. The resulting mixture was stirred atroom temperature for 2 h. LCMS indicated the reaction was completed. The solvent was removed in vacuum. The water phase was extracted with EA (10 ml*3), the combined organic layer was washed with water (10 mL), brine (10 mL), dried overanhydrous sodium sulfate and concentrated to dryness under vacuum. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1%FA), 10% to 100% gradient in 30 min; detector, UV 254 nm to give tert-butyl N-{2-[2-(4-bromo-2-chlorophen (2 g, 67%) as a yellow oil. LCMS (ES, m / z . 392 [M+H]+.

[0572] Step 7: To a solution of tert-butyl N-{2-[2-(4-bromo-2-chlorophenyl)ethoxy]ethyl}-N- methylcarbamate (1.99 g, 5.07 mmol, 1.00 equiv) in DMF (20 mL) were added zinc cyanide (713.99 mg, 6.08 mmol, 1.2 equiv) and Pd(PPh3)4 (586 mg, 0.51 mmol, 0.1 equiv) under N2 at room temperature. The resulting mixture was stirred at 80 °C for 16 h. LCMS indicated the reaction was completed. After cooled to room temperature. The resulting mixture was diluted with water (200 mL), extracted with EA (100 mL*3),The combined organic layer was washed with water (100 mL), brine (100 mL), dried over anhydrous sodium sulfate and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =2: 1) to give tert-butyl N-{2-[2-(2-chloro-4-cyanophenyl)ethoxy]ethyl}-N-methylcarbamate (1.62 g,94%) of the product as a yellow oil. LCMS (ES, m / z): 339 [M+H]+

[0573] Step 8: To a solution of tert-butyl N-{2-[2-(2-chloro-4-cyanophenyl)ethoxy]ethyl}-N- methyl carbamate (1.5 g, 4.43 mmol, 1.00 equiv) in THF (15 mL) was added bromo(methyl)magnesium (2.08 mL, 55.89 mmol, 12.61 equiv) under N2 at room temperature. The resulting mixture was stirred at 50 °C for 2 h. LCMS indicated the reaction was completed. After cooled to room temperature. The reaction was quenched with 1 N HC1 (15 mL) in ice-bath. The resulting mixture was diluted with water (100 mL), extracted with EA (50 mL*3),the combined organic layer was washed with brine , dried over anhydrous sodium sulfate and concentrated to dryness. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =2: 1) to give tert-butyl (2-(2-chloro-4-cyanophenethoxy)ethyl)(methyl)carbamate (1.06 g, 59%) of the product as a yellow oil LCMS (ES, m / z . 356 [M+H]+

[0574] Step 9: To a solution of tert-butyl N-{2-[2-(4-acetyl-2-chlorophenyl)ethoxy]ethyl}-N- methylcarbamate (500 mg, 1.41 mmol, 1.00 equiv)*2 in Pyridine (5 mL)*2 was added SeO2 (312 mg, 2.81 mmol, 2 equiv)*2 at room temperature under N2. The resulting mixture was stirred at 100 °C for 3 h. After cooled to room temperature, DCM (20 mL)*2 was added. The resulting mixture was filtered. After he filtrate was cooled to 0-5 °C, methyl chloroformate (1195 mg, 12.65 mmol, 9 equiv)*2 was added. The resulting mixture was stirred at 0-5 °C for 16 h. LCMS indicate the reaction was completed. The resulting mixture was diluted with DCM (100 mL) washed with 1 NHC1 (240 mL), aqueous bicarbonate sodium (200 mL), water (100 mL), brine (100 mL), dried over anhydrous sodium sulfate and concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluted with (PE: EtOAc =1 :3) to give methyl 2-[4-(2-{2-[(tert- butoxycarbonyl)(methyl)amino]ethoxy}ethyl)-3-chlorophenyl]-2-oxoacetate (770 mg, 65%) as a yellow oil. LCMS (ES, m / zy 400 [M+H]+

[0575] Step 10: DAST (8 mL) was added into methyl 2-[4-(2-{2-[(tert- butoxycarbonyl)(methyl)amino]ethoxy}ethyl)-3-chlorophenyl]-2-oxoacetate (750 mg, 1.88 mmol, 1.00 equiv) at room temperature. The resulting mixture was stirred at room temperature for 3 h. LCMS indicated the reaction was completed. The resulting mixture was diluted with DCM (50 mL) and neutralized with a.q. NaHCOs to pH to 7, The organic layer was separated out, The water phase was extracted with DCM (100 mL). The combined organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous sodium sulfate and concentrated to dryness under vacuum. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1%FA), 0% to 60% gradient in 30 min; detector, UV 220 nm to give methyl 2-[4-(2-{2-[(tert- butoxycarbonyl)(methyl)amino]ethoxy}ethyl)-3-chlorophenyl]-2,2-difluoroacetate (550 mg, 68%) as a yellow oil. LCMS (ES, m / zy 422 [M+H]+.

[0576] Step 11 : To a solution of methyl 2-[4-(2-{2-[(tert- butoxycarbonyl)(methyl)amino]ethoxy }ethyl)-3-chlorophenyl]-2,2-difluoroacetate (540 mg, 1.28 mmol, 1.00 equiv) in THF (10 mL) was added a solution of LiOH.H2O (107.43 mg, 2.56 mmol, 2 equiv) in H2O (5 mL) at room temperature. The resulting mixture was stirred at room temperature, for 2 h. LCMS indicated the reaction was completed. The solvent was removed under vacuum. The water was diluted with water (10 mL), adjusted to PH to 3-4 with IN HC1, extracted with EA (10 mL*3),the combined organic layer was washed with brine (10 mL), dried over anhydrous sodium sulfate and concentrated to dryness under vacuum to give [4-(2-{2-[(tert- butoxycarbonyl)(methyl)amino]ethoxy}ethyl)-3-chlorophenyl]difluoroacetic acid (500 mg, 82%) as a yellow oil. LCMS (ES, m zy 408 [M+H]+

[0577] Step 12: To a solution of [4-(2-{2-[(tert-butoxycarbonyl)(methyl)amino]ethoxy}ethyl)- 3-chlorophenyl]difluoroacetic acid (490 mg, 1.21 mmol, 1.00 equiv) in DMF (5 mL) were added HATU (685 mg, 1.80 mmol, 1.5 equiv), 3-[5-(aminomethyl)-l-oxo-3H-isoindol-2-yl]piperidine- 2, 6-dione, hydrochloried (448 mg, 1.41 mmol, 1.2 equiv) and DIE A (621 mg, 4.80 mmol, 4.0 equiv) at oom temperature in air. The resulting mixture was stirred at room temperature for 2 h. LCMSindicated the reaction was completed. The reaction was diluted with water (50 mL), The resulting mixture was extracted with EA (20 mL*3).The combined organic layer was washed with brine (10 mL), dried over anhydrous sodium sulfate and concentrated to dryness under vacuum. The residue was purified by reverse flash chromatography with the following conditions: column, Cl 8 silica gel; mobile phase, ACN in water(0.1%FA), 0% to 60% gradient in 30 min; detector, UV 254 nm to give tert-butyl N-[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5- yl]methyl}carbamoyl)difluoromethyl]phenyl}ethoxy)ethyl]-N-methylcarbamate (305 mg 35%)as a pale yellow solid. LCMS (ES, m / z): 663[M+H]+&563[M+H-100]+&607[M+H-56]+

[0578] Step 13: To a stirred solution of tert-butyl N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5-yl]methyl]carbamoyl)difluoromethyl]phenyl]ethoxy)ethyl]-N- methylcarbamate(170 mg, 0.26 mmol, 1.00 equiv) in DCM(5 mL) was added TFA(1 mL,) dropwise at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. LCMS indicated the reaction was completed. The resulting mixture was concentrated under reduce pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, 0.05%FA in water, 5% to 80% gradient in 30 min; detector, UV 254,220 nm.to afford 2-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-N-[[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5-yl]methyl]-2,2-difhioroacetamide (120 mg, 83.14%) as a white solid. LCMS (ES, m / z):564 [M+H]+

[0579] Step 14: To a solution of 2-(3-chloro-4-{2-[2-(methylamino)ethoxy]ethyl}phenyl)-N- {[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5-yl]methyl}-2,2-difluoroacetamide; trifluoroacetic acid (86 mg, 0.097 mmol, 1.00 equiv) in DMF (2 ml) were added DIEA (62 mg, 0.49 mmol, 5 equiv) and methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert- butoxycarbonyl)amino]ethyl}carbamoyl)-4-{[(4- nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxane-2-carboxylate (76 mg, 0.10 mmol, 1 equiv) at room temperature. The reaction was stirred at room temperature for 16 h. LCMS indicated the reaction was completed. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water(0.05%TFA), 0% to 60% gradient in 30 min; detector, UV 254 nm to give methyl (2S,3S,4S,5R,6S)-3,4,5- tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-[({[2-(2-{2-chloro-4- [({[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5-yl]methyl}carbamoyl)difluoromethyl]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phen oxy]oxane-2-carboxylate (110 mg, 88% )as a yellow solid. LCMS (ES, m z 1215 [M+H]+

[0580] Step 15: A solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert- butoxycarbonyl)amino]ethyl}carbamoyl)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)- l-oxo-3H-isoindol-5- yl]methyl}carbamoyl)difluoromethyl]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phen oxy]oxane-2-carboxylate (100 mg, 0.08 mmol, 1.00 equiv) in THF (5 mL) / HCl(6M) (5 mL) was stirred at 45 °C for 3 h. LCMS indicated the reaction was completed. The solvent was removed under vacuum and the residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water(0.1%FA), 0% to 40 % gradient in 30 min; detector, UV 254 nm to give (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2- (2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5- yl]methyl}carbamoyl)difluoromethyl]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phen oxy}-3,4,5-trihydroxyoxane-2-carboxylic acid ( 24 mg, 25%) as a white solid. LCMS (ES, m / z): 975 [M+H]+

[0581] Step 16: To a solution of (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2-(2- {2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H-isoindol-5- yl]methyl}carbamoyl)difluoromethyl]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phen oxy }-3,4,5-trihydroxyoxane-2-carboxylic acid (20 mg, 0.02 mmol, 1.00 equiv) in DMF (ImL) was added 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxopyrrol-l-yl)hexanoate (6 mg, 0.02 mmol, 1.00 equiv) and DIEA(13 mg, 0.10 mmol, 5.00 equiv) at room temperature in air. The resulting mixture was stirred at room temoeratrue for Ih. The reaction mixture was purified by reverse flash chromatography with the following conditions: Column: XBridge Shield RP18 OBD Column, 30*150 mm, 5um; Mobile Phase A: Water(0.05%TFA ), Mobile Phase B: ACN; Flow rate: 60 mL / min; Gradient: 21% B to 51% B in 7 min, 51% B; Wave Length: 254 nm; RTl(min): 6.07 to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-l-oxo-3H- isoindol-5- yl]methyl}carbamoyl)difluoromethyl]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2- ({2-[6-(2,5-dioxopyrrol-l-yl)hexanamido]ethyl}carbamoyl)phenoxy}-3,4,5-trihydroxyoxane-2- carboxylic acid; trifluoroacetic acid (10.9 mg, 37%) o as a white solid. LCMS (ES, m / z): 1168 [M+H]+; ’H NMR (400 MHz, DMSO-tL) 5 12.90 (br, IH), 10.99 (s, IH), 9.68 (t,J=6Hz, IH), 8.28 (t / =5.6Hz IH), 7.49 (t,J=5.2Hz, IH), 7.77 (s , IH), 7.69-7.67 (m, IH), 7.59 (s, IH), 7.55-7.43 (m,4H), 7.38-7.36 (m, 1H), 7.25-7.21 (m, 1H), 7.08-6.95 (m, 2H), 5.80-5.40 (m, 2H), 5.12-5.08 (m, 2H), 5.01 (s, 2H), 4.46-4.40 (m, 3H), 4.31-4.27 (m, 1H), 3.98-3.96 (m, 1H), 3.63-3.62 (m, 3H), 3.5 (s, 2H), 3.41-4.31 (m, 8H), 3.37-3.18 (m, 2H), 2.98-2.90 (m, 3H), 2.79 (d / =14.4, 3H), 2.67-2.58 (m, 1H), 2.40-2.33 (m, 1H), 2.06-1.99 (m, 3H), 1.48-1.43 (m, 4H), 1.18-1.14 (m, 2H).

[0582] Compound (1-9) was prepared using the procedure described in WO2023 / 037268, which is incorporated herein by reference in its entirety.Example 1-10: Synthesis of Compound (I- 10)

[0583] Compound (I- 10) was prepared using the procedure described in WO2021 / 198965, which is incorporated herein by reference in its entirety.Example 1-11: Synthesis of Compound (I- 11)

[0584] Compound (1-11) was prepared using the procedure described in WO2022 / 254376, which is incorporated herein by reference in its entirety.Example 1-12: Synthesis of Compound (1-12)

[0585] Compound (1-12) was prepared using the procedure described in WO2021 / 198965 which is incorporated herein by reference in its entirety.Example 1-13: Synthesis of Compound (1-13)

[0586] Compound (1-13) was prepared using the procedure described in WO2021 / 198965 which is incorporated herein by reference in its entirety.Example 2: Anti-CD123 Antibody Purification

[0587] Anti-CD123 antibodies were expressed in CH0-K1 cells and purified.

[0588] The following antibodies were expressed using an IgGl N297A constant region (SEQ ID NO:51): 32703 (N297A), CD123.001, CD123.002, CD123.022, CD123.023, CD123.024, CD123.025, CD123.026, CD123.027, CD123.028, CD123.029, CD123.030, CD123.031, CD123.032, CD123.033, CD123.034, CD123.035, and CD123.036.

[0589] The following antibodies were expressed using an IgGl L234A L235A N297A constant region (SEQ ID NO: 53): 32703 (N297A L234A L235A), CD123.003, CD123.004, CD123.005,CD123.006, CD123.007, CD123.008, CD123.009, CD123.010, CD123.011, CD123.012,CD123.013, CD123.014, CD123.015, CD123.016, CD123.017, CD123.018, CD123.019,CD123.020, and CD123.021.

[0590] Supernatants of transfected CH0-K1 cells were harvested by centrifugation and FPLC- based affinity purification using Protein A MabSelect SuRe (Cytiva, Cat. #11003494) chromatography column, followed by washing with PBS. Antibodies were eluted with 0.1 M Glycine pH 3.0 and neutralized with 1 M Tris pH 9.0. Buffer exchange with the final formulation buffer (20 mM Histidine, 250 mM Sucrose, pH 6.5) was performed using Amicon Ultra-4 Centrifugal Filters (Merck Millipore).Example 3 : Preparation and Characterization of GSPT1 Neodegrader Conjugate

[0591] 5 -50 mgs of the antibody in (20 mM histidine, 250 mM sucrose pH 6.5) were mixed with the conjugation buffer (PBS pH 7.4) to bring the antibody concentration in the reaction to 6 mg / mL. Then it was reacted with (tris(2-carboxyethyl)phosphine) (“TCEP,” 5 mM, 2.0-2.5 eq.) for 2 h at 37 °C or for 16 h at 22 °C. Then PBS pH 7.4, DMA, and Linker Payload (10 mM in DMA, 7 eq.) were sequentially added to bring the antibody concentration to 5.4 mg / mL and the % DMA to 10 %. Then the mixture was allowed to react for 1 h at 22 °C. The resulting conjugate was purified and concentrated with Amicon (3 OK, 20DV) with 20 mM succinate, 8% sucrose, pH 5.5 buffer. The conjugate was then sterile filtered through 0.22 pm PVDF syringe filters to get the final purified conjugate with the specifications described in Table 1. After characterization, 0.01% tween-20 was added to the conjugate to get the final product in formulation buffer: 20 mM succinate, 8% sucrose, 0.01% tween, pH 5.5.

[0592] Conjugate concentration was measured by UV-Vis against a calibration curve. Monomer ratio was measured by SEC-HPLC. Free drug ratio was measured by HISEP assay where the area under the curve was used to determine concentration of the free drug using a standard curve which was obtained for the payload. DAR was determined by reducing RPLC-MS or Native SEC -MS.Preparation of Conjugate C

[0593] To the 50 mL EP tube containing 19.5 mg of antibody CD123.001 was added conjugation buffer (PBS, pH7.4) and 5 mM TCEP solution (2.4 equiv.). The antibody concentration in the reaction was 6 mg / mL. The reactions were mixed well and incubated at 22 °C for 16 hrs. AddedDMA and stock 10 mM Compound (I) in DMA solution (7 equiv.) into the reactions, mixed well and incubated at 22 oC for another 1 hour. The antibody concentration during conjugation was 5.4 mg / mL. The crude conjugation was purified with Amicon (30K, 20DV) with 20 mM succinate, 8% sucrose, pH 5.5 buffer. The fractions were collected and filtered with 0.22 pm filter. After final characterization, 0.01% PS20 was added to the conjugates and mixed well end over end. The purified conjugate was found to have an average DAR of 3.7 by reduced LCMS method, the SEC- HPLC analysis result showed monomer ratio of 97.47% and the yield was 98%.Table 1 : Specifications of conjugates via described bioconjugation procedures

[0594] In addition, Conjugate G was made by linking Compound (I) to Synagis, which bind to RSV virus.Preparation of Conjugate H

[0595] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg / mL, 0.450 mL) was treated with 5 mmol / L (aq) TCEP solution (13.621 uL, 0.068 pM, 10 eqv) and incubated at 37 °C for 2 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column and the volume was reduced to 580 uL. To the reduced colume was added 100 uL of additional buffer solution followed by 99.6 uL of fresh DMA and 10 mM of Compound (I) in DMA (20.4 uL, 0.204 mM, 12 eqv) in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal FilterlO KDa. The purified conjugates was found to have an average DAR of 8 by HIC and LC-MS, the SEC-HPLC analysis result showed monomer ratio of 98.1% and the yield was 44%.Preparation of Conjugate I

[0596] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-1) in DMA in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LC-MS, the SEC-HPLC analysis result showed monomer ratio of 97.2% and the yield was 58%.

[0597] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-2) in DMA in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LC-MS, the SEC-HPLC analysis result showed monomer ratio of 98.7% and the yield was 50%.Preparation of Conjugate K

[0598] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-3) in DMA in such a way that final ratio of DMA:aqueos buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LC-MS, the SEC-HPLC analysis result showed monomer ratio of 97.2% and the yield was 70%.Preparation of Conjugate L

[0599] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of linker-payload as 10 mM Compound (1-4) in DMA in such a way that final ratio of DMA: aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween- 20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LC-MS and the SEC-HPLC analysis result showed monomer ratio of 97.0% and the yield was 53%.Preparation of Conjugate M

[0600] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-5) in DMA in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LCMS and the SEC-HPLC analysis result showed monomer ratio of 99.9% and the yield was 50%.Preparation of Conjugate N

[0601] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-6) in DMA in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LCMS and the SEC-HPLC analysis result showed monomer ratio of 100% and the yield was 57%.Preparation of Conjugate O

[0602] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-7) DMA in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LCMS the SEC-HPLC analysis result showed monomer ratio of 100% and the yield was 44%.Preparation of Conjugate P

[0604] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-8) in DMA in such a way that final ratio of DMA:aqueos buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LCMS the SEC-HPLC analysis result showed monomer ratio of 98.8% and the yield was 55%.

[0605] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (1-9) in DMA in such a way that final ratio of DMA:aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LCMS and the SEC-HPLC analysis result showed monomer ratio of 99.4% and the yield was 67%.

[0607] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (2 mg) was treated with 10 eqv of 5 mmol / L (aq) TCEP solution and incubated at 37 °C for 1 h to fully reduce the interchain disulfide bonds. The reduced antibody was purified into 50 mM EPPS, 5 mM EDTA pH 7.0 by gel filtration using a Zeba 40K spin column. To the solution of reduced antibody was added additional buffer solution followed by fresh DMA and 12 eqv of 10 mM Compound (I- 10) in DMA in such a way that final ratio of DMA: aqueous buffer solution was 15:85 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC and LCMS the SEC-HPLC analysis result showed monomer ratio of 97.0% and the yield was 47%.I l lPreparation of Conjugate S

[0608] In a 1.5 mL centrifuge tube, mAb CD123.001 in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer (1 mg) was added additional buffer solution followed by fresh DMA and 15 eqv of 10 mM Compound 1-11 in DMA in such a way that final ratio of DMA: aqueous buffer solution was 20:80 v / v. The resulting mixture was incubated at 25 to 27 °C for 2 hours in a dry bath. The resulting conjugate was purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K column followed by dialysis against the formulation buffer using a Amicon ultra centrifugal Filter 10 KDa. The purified conjugates were found to have an average DAR of 8 by HIC the SEC-HPLC analysis result showed monomer ratio of 97.7% and the yield was 63%.Preparation of Conjugate T

[0609] In a 1.5 mL centrifuge tube, mAb CD123.001 (2 mg, 6.5 mg / mL) in 50 mM EPPS, 5 mM EDTA, pH 7.4 buffer was reduced by adding 10 equivalents of TCEP (27.3 pL of 5 mM solution in sterile water). The reaction mixture (400 pL total volume) was incubated at 37 °C in a dry bath for 2 hours, then cooled to room temperature. The reduced antibody was subsequently purified by gel filtration using a Zeba 40K 2 mL spin column equilibrated with the same EPPS buffer. To 290uL of the reduced antibody, EPPS buffer and fresh DMA were added to achieve a final DMA:buffer ratio of 20:80 (v / v), followed by the addition of Compound 1-13 (10 mM in DMA, 15 equivalents). The mixture was briefly centrifuged, then incubated at room temperature (25-27 °C) for 2 hours. Then the reaction mixtures were purified into 20 mM succinate, 8% sucrose, 0.01% Tween-20, pH 5.5 formulation buffer by gel filtration using Zeba 40K 2 mL spin columns, followed by concentration using VivaSpin centrifugal filter units. The purified conjugate was found to havean average DAR of 8 by HIC the SEC-HPLC analysis result showed monomer ratio of 97.0% and the yield was 47%.Preparation of Conjugate U

[0610] In a 1.5 mL centrifuge tube, mAb CD123.001 (2 mg, 5.8 mg / mL) in 50 mM EPPS, 5 mM EDTA, pH 7.0 buffer was reduced by adding 10 equivalents of TCEP (27.3 pL of 5 mM solution in sterile water). The 400 pL reaction mixture was incubated at 37 °C in a dry bath for 2 hours, then allowed to cool to room temperature. The reduced antibody was purified using a Zeba 40K 2 mL spin column equilibrated with EPPS buffer, resulting in a final volume of 580 pL. Into 290uL of the reduced antibody stock solution, 50 pL of EPPS buffer and DMA were added to achieve a final DMA content of 15% (v / v), followed by 15 equivalents of Compound 1-12 (10 mM solutions in DMA). The mixture was briefly centrifuged, mixed by pipetting, and incubated at room temperature (25-27 °C) for 2 hours. The mixture was then purified by gel filtration using Zeba 40K 2 mL spin columns. The resulting antibody-drug conjugate was further concentrated using VivaSpin centrifugal filter units. The purified conjugate was found to have an average DAR of 8 by HIC the SEC-HPLC analysis result showed monomer ratio of 99.4% and the yield was 55%.Example 4: Binding Characterization of Anti-CD 123 AntibodiesTarget Binding ELISA

[0611] To evaluate the target binding activity of anti-CD123 antibodies binding to human CD 123 protein was performed by an ELISA assay. ELISA plates were coated with recombinant human CD123 protein (Acrobiosystems, Cat.#ILA-H52H6) and incubated overnight at 4°C. The plates were washed four times with washing solution (0.05% tween-20 in PBS, pH 7.4). The plates were blocked with 300 pL of 3% skim milk and incubated for 2 hrs at room temperature. After washing as above, several concentrations (10 pg / mL - 0.03 ng / mL) of antibody samples in PBS, pH 7.4 were incubated for 1 hr at room temperature. After washing as above, bound samples were detected by labeling with an HRP-conjugated anti-human IgG (H+L) antibody (Jackson ImmunoResearch Labs, Cat.#109-035-088) and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50- tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader. The binding curve was fitted using a 4-parameter sigmoidal in a nonlinear regression equation using Prism 9.0.2. Under this experimental condition, FIG. 1Ademonstrated that h7G3 (see International Patent Publication No. W02016 / 201065 at SEQ ID NOs: 1 and 2) and 32703 showed similar ECso values, and both showed better binding than Pivekimab (see US Patent No. 10,077,313 at SEQ ID NO:34 or 35; “Pivekimab,” as used herein, refers to an antibody with an IgGl WT Fc unless expressly indicated otherwise).Species Cross-Reactivity

[0612] To evaluate cross-reactivity of anti-CD123 antibodies with cynomolgus monkey and mouse CD 123, the binding activity of antibodies to CD 123 proteins from different species consisting of cynomolgus monkey and mouse were measured by indirect enzyme-linked immunosorbent assay (ELISA) to determine cross-reactivity.

[0613] The Immuno Clear Standard Module Plates were coated overnight at 4 °C with 100 pL of 2 pg / mL of recombinant CD123 protein (cynomolgus monkey Acrobio, Cat.# ILA-C52H6 or mouse Acrobio, Cat.# ILA-M52H4) and blocked with a blocking solution (3% skim milk in PBS, pH 7.4) for 2 h at room temperature. After washing 4-times with washing solution (0.05% tween- 20 in PBS, pH 7.4), several concentrations (5-fold dilution 8 points at 10 pg / mL) of anti-CD123 antibodies in PBS, pH 7.4 were incubated for 1 h at room temperature. After washing 4-times with a washing solution, bound antibodies were detected by HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch Labs, Cat.#109-035-088) and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader. The standard curve was fitted to a 4-parameter logistic equation using SoftMax Pro 7.1. FIG. IB shows that anti-CD123 antibodies h7G3 and 32703 bind to recombinant cynomolgus monkey CD123, but not to mouse CD123.Cell Surface Binding

[0614] The cellular binding of each anti-CD123 antibody was tested in the CD 123 positive acute myeloid leukemia (AML), MV4-11 cell line. Cells were harvested and washed twice with incubation buffer (2% FBS / PBS). Cells were seeded at approximately 1 x 105cells / well in a roundbottom 96-well plate. The cells were incubated with various concentrations of anti-CD123 antibodies at 4 °C for 1 h. Maximum final antibody concentration is 100 nM, with a 4-fold dilution series for a total of 7 dilutions (100 to 0.024414 nM). After cells were washed twice with incubationbuffer, the cells were incubated with Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermofisher, # A-l 1013) at 4 °C for 1 h. The cells were washed twice and resuspended with incubation buffer. The fluorescence signal was detected by flow cytometry. The results shown in FIG. 2 demonstrate that cell surface binding of all anti-CD123 antibodies on MV4-11 cells was dose-dependent and that the antibodies exhibited essentially similar binding properties.Target Binding Comparison Between Naked Antibody and Conjugate

[0615] To compare the target binding of naked antibody (32703 antibody) and conjugate, the binding activity was analyzed by ELISA method. The Immuno Clear Standard Module Plates were coated overnight at 4 °C with 100 pL of 2 pg / mL of recombinant human CD 123 protein and blocked with a blocking solution (3% skim milk in PBS, pH 7.4) for 2 h at room temperature. After washing 4-times with washing solution (0.05% Tween-20 in PBS, pH 7.4), several concentrations (4-fold dilution 8 points at 10 pg / mL) of anti-CD123 antibodies in PBS, pH 7.4 were incubated for 1 h at room temperature. After washing 4-times with a washing solution, bound antibody or conjugate was detected by HRP-conjugated goat anti-human IgG (H+L) antibody and subsequent incubation with 100 pL of ultra- TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader. The binding curve was fitted by 4-parameters sigmoidal in non-linear regression equation among XY analyses using Prism 9.0.2. FIG. 3 demonstrates that the CD123 binding of ADC was similar to that of naked 32703 antibody.Example 5: Evaluation of in vitro cytotoxicity of 32703 conjugates

[0616] An in vitro cytotoxicity assay was performed to demonstrate the cytotoxic activity of chimeric 32703 antibody conjugate with Compound (I). Test articles were Conjugate A, the 32703 antibody, GSPT1 Neodegrader (XI), and CC-885 (Celgene) and CC-90009 (Celgene) for MV4-11 or Conjugate G for TOM-1 as non-binding control. Their cytotoxicity was evaluated by using two human leukemia cell line, MV4-11 which is AML cell line and TOM-1 which is B-ALL cell line. Both cell lines are known to express CD123. MV4-11 cells and TOM-1 cells were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of were Conjugate A, the 32703 antibody, GSPT1 Neodegrader (XI), Conjugate G, CC-90009, andCC-885 for 72 hrs. The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay that is colorimetric assay (Dojindo Molecular Technologies, Inc). The absorbance at 450 nm was read using the SpectraMax M5e plate reader. The normalized percentage values were calculated by dividing each treated sample value by the untreated cells and IC50 values were calculated using the Prism software. As shown in the Table 2, FIG. 4A, and FIG. 4B, it was observed that Conjugate A, which was a 32703 antibody conjugate, had cytotoxic activity in both cell line, MV4-11 and TOM-1, and it showed a more potent cytotoxicity than other test articles. Conjugate A was showed more potent around 1,000 times than GSPT1 Neodegrader (XI), which was the neodegrader of Conjugate A.Table 2: In vitro efficacy of 32703 conjugatesExample 6: In Vivo Anti-Tumor Efficacy of Conjugate

[0617] The purpose of this study was to test initial efficacy in a model of disseminated AML and compare head-to-head with anti-CD123 antibodies conjugated to Compound (I). Immunocompromised mice of strain NCG were inoculated with the human AML cell line MV4- 11-LUC in the tail vein. Two weeks post inoculation, mice were staged by imaging for the amount of Bioluminescence (BLI), which is proportional to the number of MV4-11 LUC cells in the animal. Mice with equivalent levels of BLI were then randomized into 5 groups: 1) Vehicle control; 2) Conjugate A at 0.5 mg / kg; 3) Conjugate A at 5 mg / kg; 4) Conjugate B at 0.5 mg / kg; 5) Conjugate B at 5 mg / kg. All test articles were administered as a single dose by I.V. injection. BLI and body weight were measured for 60 days following treatment.

[0618] As shown FIG. 5, by quantification, Conjugate A at 5 mg / kg led to a 99.9% reduction in AML burden by BLI compare vehicle control (p=0.0002). Looking at the other groups: Conjugate A at 0.5 mg / kg, Conjugate B at 0.5 mg / kg, 5 mg / kg produced significant antitumor activities with 93.4%, 69.7% and 93.9% reduction of BLI signal compared with vehicle control group (P values < 0.01), respectively.

[0619] With respect to the survival times of animals in different groups, Conjugate A at 0.5 mg / kg led to a survival benefit of 63% increased life span versus vehicle control (p<0.001). In terms of the other groups, Conjugate A 5 mg / kg and Conjugate B at 0.5 mg / kg also significantly prolonged animals’ survival times compared with vehicle control group (P values < 0.01).

[0620] Regarding the safety profile, some animals treated with Conjugate A and Conjugate B at 5 mg / kg dose levels were found dead with low tumor BLI signals, that might be due to the individual differences in response to drug of mice. In the later stage of the study, the obvious bodyweight loss and death of animals in vehicle control group are most likely due to the systemic tumor burden in mice (FIG. 6).Example 7: CDR Engineering of 32703 Antibodies and Evaluation of Variants

[0621] mAbs are usually further engineered to meet the requirements of the project, such as humanization, affinity maturation, Fc-engineering, etc. In this stage, in addition to monitoring for appearance, expression level and purity, PTM hotspots with high risk should be removed and thermal stability should be performed. High-risk motifs, including NG / NS / DG motifs, extra Cys residue and N-glycosylation motif on variable regions, should be removed to mitigate potential risks to on efficacy and safety.SDS-PAGE Analysis of CDR Mutants

[0622] One potential N-glycosylation site Asn26 (Asn-X-Ser / Thr) was recognized in VL CDR of the parental 32703 antibody. So further engineering was proceed to create a potential variant that can remove an existing N-linked carbohydrate chain. Three CDR engineered clones were produced and evaluated considering the amino acid properties and germline frequencies. FIG. 7 is a SDS-PAGE gel analysis result showing variants including CDR wild-type antibody under reducing and non-reducing conditions gave different banding patterns. The CDR wild-type clones (32703(N297A L234A L235A) and 32703) showed two LC bands, whereas the CDR engineered clones (CD123.003, CD123.004, and CD123.005) showed one LC band under reducing condition, which verified that existing N-glycosylation site was successfully removed.Target Binding Analysis of CDR Mutants

[0623] Binding to human CD 123 protein was performed by an ELISA assay to determine if substitution of one amino acid in the CDR site will affect the target binding property. ELISA plateswere coated with recombinant human CD 123 protein (Acrobiosy stems, Cat.# ILA-H52H6) and incubated overnight at 4 °C. The plates were washed four times with washing solution (0.05% tween-20 in PBS, pH 7.4). The plates were blocked with 300 pL of 3% skim milk and incubated for 2 hrs at room temperature. After washing as above, several concentrations (5-fold dilution 8 points at 10 pg / mL) of antibody samples in PBS, pH 7.4 were incubated for 1 h at room temperature. After washing as above, bound samples were detected by labeling with an HRP- conjugated anti-human IgG (H+L) antibody (Invitrogen, Cat.#A18811) and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader. The binding curve was fitted using a 4-parameter sigmoidal in a non-linear regression equation using Prism 9.0.2. FIG. 8 indicates that the position of N-glycosylation on the light chain CDR site has no impact on antigenantibody binding.Comparison of Internalization Kinetics of 32703 CDR Mutants

[0624] To compare cellular internalization kinetics of anti-CD123 antibodies including CDR mutant (CD123.003), the pHAb labeled antibodies were used to perform internalization assay. pHAb is a pH-dependent dye, characterized by high fluorescence at acidic pH changes. Since pHAb-labeled antibodies do not fluoresce at neutral pH on the cell surface, a high fluorescence signal indicates that the antibody has been internalized (Promega, Inc).

[0625] MV4-11 cells were harvested and washed twice with binding buffer (2% FBS / PBS). Prepared 1.5 x 105cells / mL approximately with of binding buffer. The cells were incubated with 500 nM of the pHAb labeled antibody at 37 °C for 1, 6, or 24 hrs. After wash twice with cold PBS, the cells were resuspended with cold binding buffer. The signal was detected by flow cytometer. The plots showed the mean fluorescence intensity (MFI) compared with the incubation time. FIG. 9 showed the internalization kinetic of tested anti-CD123 antibodies were comparable.Surface Plasmon Resonance

[0626] The target binding affinities of 32703 and 32703 antibody variants (CD123.003 and 32703 (N297A)) were determined by surface plasmon resonance technology using Biacore T200. Amine coupling kit (BR100050), human antibody capture kit (BR100839), Sensor chip CM5 (BRI 00012) and HBS-EP+ Buffer 10x (BRI 00669) are purchased from GE healthcare. Accordingto the kit instructions, use 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride (1- Ethyl-3 -(3 -dimethylaminopropyl)carbodiimide hydrochloride, EDC) and N-hydroxyl Succinimide (N-Hydroxysuccinimide, NHS) activates the surface of the carboxylated CM5 chip.Dilute the anti-human IgG (Fc) antibody (capture antibody) to 25 pg / mL in 10 mM Sodium acetate pH 5.0, and then perform immobilization at 25 °C using a flow rate of 10 pL / min for 6 minutes to achieve immobilization levels approximately 10,000 response units (RU).

[0627] After the capture antibody was injected, 1 M ethanolamine was injected for 7 minutes to deactivation. For kinetic measurement, dilute the anti-CD123 antibody in HBS-EP buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (“EDTA”), and 0.005% (v / v) Surfactant P-20) to 0.5 pg / mL and inject 10 pL / min to ensure that about 100 RU of the antibody is captured by the anti-human antibody. Subsequently, serially diluted recombinant human CD123 protein to various concentrations (50-1.5625 nM) in HBS-EP buffer, was injected over immobilized antibodies at a flow rate of 30 pL / min for 3 min with 6 min dissociation per cycle. After each cycle, surfaces were regenerated with Regeneration Buffer (3 M MgCh) for 1 min. Dissociation (kOff) and association (kon) rate constants were obtained by non-linear regression analysis of the primary sensorgram data according to a 1 : 1 binding Langmuir model using the BIAevaluation software provided by manufacturer. The dissociation constant (KD) was calculated using the formula, KD=kOff / kOn. The fitting results in Table 3 showed that all tested 32703 antibody variants have similar binding parameters, including the binding kinetics results of N-glycosylation site engineered antibody.Table 3: Target binding affinity parameters of 32703 antibody variants for rhCD123 proteinExample 8: Additional CDR Engineering of 32703 Antibody

[0628] To mitigate potential risks to efficacy and safety as described above, we analyzed the various chemical modification potential sites present in CDRs of CD 123.003. As a result ofprimary sequence analysis, several sequence liabilities were identified. To minimize the chance of unfavorable developability issue, sequences containing undesirable liabilities such as asparagine deamidation and aspartate isomerization were substituted with amino acid(s) with similar property or structure as shown in the Table 4, with multiple combinations to generate antibody variants.Table 4: Description of additional CDR mutants

[0629] With reference to Example 6, the target binding affinity of the additional CDR variants was measured with Biacore T200, and the results are shown in Table 5. This result demonstrates that mutations D99E in VH and N28S / N30K in VL do not affect target binding.Table 5: Binding parameters of additional CDR mutantsExample 9: Humanization of 32703 Antibody

[0630] Humanization of the murine anti-human CD123 antibody, 32703 (originally mouse reagent Ab), was carried out in a series of site modification steps. Human germline frameworks having the highest overall homology to the original murine sequences were selected three for each heavy chain and light chain antibody sequence to provide the framework for CDR-grafting purposes. Mouse 32703 mAb CDRs grafted onto human frameworks produced are those listed in Table 6.Table 6: Description of human frameworks for each humanized variant

[0631] With reference to Example 6, the binding affinity of the humanized variants was measured with Biacore T200, and the results are shown in Table 7. SPR analysis confirmed that CD123.014, CD123.016, and CD123.017 retained target binding. However, CD123.014 and CD123.017 had significantly lower expression levels during antibody production. CD123.016 was selected as a template for further optimization.Table 7: Binding parameters of humanized variants

[0632] In addition, the vernier zone residues present in the human framework were replaced with human sequences to minimize immunogenicity while maintaining the target binding of parent antibody. In the process, to maintain the target binding affinity of the parent antibody during humanization and minimize the chemical modification sites present in the CDR, the combination of human framework of CD123.016 and the CDR mutant (from Example 8, D99E in VH and N28S / N30K in VL) was used as a template introduce vernier zone back mutations (listed in Table 8) at key positions in the framework region.Table 8: Mutation site descriptions of additional humanization variants

[0633] With reference to Example 6, the binding affinity of the humanized variants was measured with Biacore T200, and the results are shown in Table 9.Table 9: Binding parameters of additional humanization variantsExample 10: Binding Characterization of Humanized 32703 AntibodySpecies Cross-Reactivity

[0634] To evaluate cross-reactivity of anti-CD123 antibodies, including humanization 32703 (CD123.001) with cynomolgus monkey and mouse CD123, the binding activity of antibodies to CD123 proteins from different species consisting of cynomolgus monkey and mouse were measured by indirect enzyme-linked immunosorbent assay (ELISA) to determine cross-reactivity.

[0635] The Immuno Clear Standard Module Plates were coated overnight at 4 °C with 100 pL of 2 pg / mL of recombinant CD123 protein (cynomolgus monkey Acrobio, Cat.# ILA-C52H6 or mouse Acrobio, Cat.# ILA-M52H4) and blocked with a blocking solution (3% skim milk in PBS, pH 7.4) for 2 hrs at room temperature. After washing 4-times with washing solution (0.05% Tween- 20 in PBS, pH 7.4), several concentrations (5-fold dilution 8 points at 10 pg / mL) of anti-CD123 antibodies in PBS, pH 7.4 were incubated for 1 hr at room temperature. After washing 4-times with a washing solution, bound antibodies was detected by HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch Labs, Cat.#109-035-088) and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450nm on a SpectraMax M5e Multi-Mode plate reader. The standard curve was fitted to a 4-parameter logistic equation using SoftMax Pro 7.1. FIG. 10A showed that all antibodies, excluding Pivekimab with an S442C mutation in the constant region (i.e., “Pivekimab(S442C)”) bind to recombinant cynomolgus monkey CD123, but not bind to mouse CD123 (FIG. 10B).Cell Surfacing Binding

[0636] To compare the cellular binding affinity of 32703(N297A) and CD123.001, the cellular binding test was performed in MV4-11 cell line. CD123 positive cell line. Specifically, cells were harvested and washed twice with incubation buffer (2% FBS / PBS). Cells were seeded at approximately 1.5 x io4cells / well in a round-bottom 96-well plate. The cells were incubated with various concentrations of anti-CD123 antibodies at 4 °C for 30 min. Maximum final antibody concentration is 100 nM, with a 3-fold dilution series for a total of 11 dilutions (100 to 0. 0.001694 nM). After cells were washed twice with incubation buffer, the cells were incubated with Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermofisher, # A-l 1013) at 4 °C for 15 min. The cells were washed twice and resuspended with incubation buffer. The fluorescence signal was detected by flow cytometry. The results showed that cell surface binding of 32703 chimeric and humanized antibodies on MV4-11 cells was dose-dependent and that the antibodies exhibited essentially similar binding properties (FIG. 11).Target Binding Comparison Between Naked Antibody and Conjugate

[0637] To compare the target binding of CD123.001 humanized antibody and conjugate, the binding activity was analyzed by ELISA method. The Immuno Clear Standard Module Plates were coated overnight at 4 °C with 100 pL of 2 pg / mL of recombinant human CD123 protein and blocked with a blocking solution (3% skim milk in PBS, pH 7.4) for 2 hr at room temperature. After washing 4-times with washing solution (0.05% Tween-20 in PBS, pH 7.4), several concentrations (5-fold dilution 8 points at 1 pg / mL) of antibody or conjugate in PBS, pH 7.4 were incubated for 1 h at room temperature. After washing 4-times with a washing solution, bound antibody or conjugate w7as detected by HRP-conjugated mouse monoclonal anti-human IgG Fc antibody and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader. The binding curve was fitted by 4-parameters sigmoidal in non-linear regression equationamong XY analyses using Prism 9.0.2. As a result, FIG. 12 showed the CD 123 binding of Conjugate C was similar to that of CD123.001.Example 11 : Competitive Binding Analysis

[0638] To evaluate the competitive binding of 32703 and other anti-CD123 antibodies (h7G3 (which contains an IgGl WT sequence), Talacotuzumab (which contains an IgGl N297A sequence), and Pivekimab(S442C)), a competitive test was performed by ELISA. The 32703(N297A) was labeled with biotin according to the method described in the manual of Thermo Scientific EZ-Link Biotin Labeling Kit to obtain biotinylated 32703(N297A).

[0639] ELISA plates were coated with recombinant human CD 123 protein (Acrobiosy stems, Cat.# ILA-H52H6) and incubated overnight at 4 °C. The plates were washed 4-times with washing solution (0.05% Tween-20 in PBS, pH 7.4). The plates were blocked with 300 pL of 3% skim milk and incubated for 2 hrs at room temperature. After washing as above, add 100 pL / well of 6 nM of biotinylated 32703(N297A) antibody (2x concentration) with anti-CD123 antibodies starting from 6000 nM (2x concentration), 10-fold, 4 points (final cone. = 3000, 300, 30, 3). Incubate the plate for 1 hr at room temperature. After washing as above, bound samples were detected by labeling with an HRP-Conjugated Streptavidin (Thermo Scientific, Cat.#N100) and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. And then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader.

[0640] As shown in FIG. 13, 32703 and Pivekimab(S442C) (the antibody portion of IMGN632) showed competitive binding against recombinant human CD123. This result indicate that the binding epitope of the 32703 antibody is similar to Pivekimab(S442C), when compare with other published anti-CD123 antibodies. However, as shown in Example 10, they are not exactly the same, as 32703 cross-reacts with CD123 on cynomolgus monkeys while Pivekimab(S442C) does notExample 12: In Vitro Cytotoxicity of Humanized 32703 Conjugate

[0641] An in vitro cytotoxicity assay was performed to compare humanized 32703 conjugates. Test articles were Conjugate C, Conjugate E, Conjugate D, Conjugate F, and CC-885 and their cytotoxicity was evaluated by using MV4-11 cell line (FIG. 14).

[0642] Cells were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of test articles for 72 hrs. The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay that is colorimetric assay (Dojindo Molecular Technologies, Inc). The absorbance at 450 nm was read using the SpectraMax M5e plate reader. The normalized percentage values were calculated by dividing each treated sample value by the untreated cells and ICso values were calculated using the Prism software.

[0643] As shown in Table 10, all conjugates showed cytotoxic activity against MV4-11, with the humanized 32703 conjugates Conjugate C and Conjugate E being more effective than Conjugate D with the parent antibody.Table 10: In vitro cytotoxicity IC50 valueExample 13: GSPT1 Degradation Activity of Humanized 32703 Conjugate

[0644] An in vitro GSPT1 degradation assay was performed to demonstrate the ability of conjugate to degrade GSPT1 via payload delivery. MV4-11 cells were seeded in 6-well culture plates. After incubating overnight, the cells were treated with serial dilution of Conjugate C, GSPT1 Neodegrader (XI), CC-885, and naked antibody (CD123.001) overnight. In particular, GSPT1 Neodegrader (XI) and CC-885 treatment concentrations were calculated taking into account the drug of antibody ratio (DAR) of Conjugate C. Protein was loaded to 4-12% NuPAGE Bis-Tris gel. Western blot with rabbit anti-GSPTl antibody (Abeam) was performed at 1 : 1000 dilution and re-blotting with P-actin HRP (Cell signaling #5125) was performed at 1 :3000 dilution. The membrane was imaged on the iBright™ FL 1500 Imaging System (Thermofisher Inc.).

[0645] As shown in FIG. 15, cells treated with Conjugate C showed dose-dependent degradation of GSPT 1 and it showed more potent degradation of GSPT 1 in low dose than GSPT I Neodegrader (XI) and CC-885. The unconjugated control, naked antibody CDI23.001 , had no effect on GSPT1 level in MV4-11 cell lines.Example 14: Antagonistic Activity Test Under IL- 3 Presence Condition

[0646] An in vitro cytotoxicity assay was performed to demonstrate the antagonistic activity of humanized 32703 antibody under IL-3 presence. Test articles were CD123.001, Talacotuzumab which had the IL-3 blocking activity, and an anti-PD-1 antibody as a non-binding control, X. Their antagonistic activity was evaluated by using a human AML cell line, MV4-11 under IL-3 presence condition. MV4-11 cells were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of CD123.001, Talacotuzumab, and the anti-PD-1 antibody for 72 hrs and 5 ng / mL of IL-3 was treated with them simultaneously. The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay that is colorimetric assay (Dojindo Molecular Technologies, Inc). The O.D450 value was read using the SpectraMax M5e plate reader. The normalized percentage values were calculated by dividing each treated sample value by the untreated cells without IL-3 and IC50 values were calculated using the Prism software.

[0647] As shown in FIG. 16, CD123.001 did not affect the cell proliferative activity under IL-3 absent condition. However, under IL-3 present condition, CD123.001 and Talacotuzumab showed to block IL-3 activity, and it was demonstrated by decreased cell proliferation. The anti-PD-1 antibody had no effect on cell proliferation under both conditions.Example 15: In Vitro Cytotoxicity of Conjugate Under IL- 3 Presence Conditions

[0648] An in vitro cytotoxicity assay was performed to demonstrate whether the efficacy of the conjugate is affected by the presence of IL-3 (FIG. 17). The test article was Conjugate C, and its efficacy was evaluated by using a human AML cell line, MV4-11 under IL-3 presence condition. MV4-11 cells were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of Conjugate C for 72 hrs and 5 ng / mL of IL-3 was treated with them simultaneously. The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay that is colorimetric assay (Dojindo Molecular Technologies, Inc). The O.D450 value was read using the SpectraMax M5e plate reader. The normalized percentage values were calculated by dividing each treated sample value by the untreated cells with or without IL-3 and IC50 values were calculated using the Prism software.

[0649] As shown in Table 11, Conjugate C showed a similar cytotoxicity activity under the presence or absence of IL-3. Therefore, IL-3 did not affect to in vitro efficacy of Conjugate C significantly.Table 11 : In vitro cytotoxicity IC50 valueExample 16: In Vivo Anti-Tumor Efficacy of Humanized 32703 Antibody Conjugate

[0650] The purpose of this study was to verify that humanization clones Conjugate C and Conjugate E did not lose in vivo efficacy as compared to Conjugate A and compared head-to-head with clinically validated anti-CD123, Conjugate F, conjugated to Compound (I). Similar to Example 6, immunocompromised mice of strain NCG were inoculated with the human AML cell line MV4-11 LUC in the tail vein. Two weeks post inoculation mice were staged by imaging for the amount of Bioluminescence (BLI) which is proportional to the number of MV4-11 LUC cells in the animal. Mice with equivalent levels of BLI were then randomized into 4 groups: 1) Vehicle control; 2) Conjugate C at 0.5 mg / kg; 3) Conjugate E at 0.5 mg / kg; 4) Conjugate F at 0.5 mg / kg. BLI and body weight were measured for 60 days following treatment.

[0651] As shown FIG. 18A and FIG. 18B, by quantification, Conjugate C at 0.5 mg / kg, demonstrated statistically significant reduction in AML. tumor burden of 99% (p < 0.0001) versus vehicle control. Survival was increased in this same group by 69% increased life span (p < 0.001) versus vehicle control (FIG. 19 and Table 12). Conjugate C performed better than clone Conjugate E and Conjugate F. Conjugate C showed similar performance to the previously tested clone - Conjugate A.Table 12: Survival curve analysis of in vivo efficacy study- Tumor Growth Delay (T-C), where T = median survival of the ADC-treated group, and C = median survival of the vehicle-treated group. % Increased Life Span (% ILS) = (T-C) / C X100%

[0652] Regarding the safety profile, some animals treated with Conjugate E at 0.5 mg / kg and Conjugate F at 0.5 mg / kg were found dead with low tumor BLI signals, that might be due to the individual differences in response to drug of mice. In the later stage of the study, the obvious bodyweight loss and death of animals in Conjugate C and vehicle control group are most likely due to the systemic tumor burden in mice (FIG. 20).Example 17: Internalization of Humanized 32703 Antibody Conjugate Compared with Other Anti-CD123 Agents

[0653] To evaluate and compare the dose-dependent internalization ability of anti-CD123 antibodies, including CD123.001, Talacotuzumab, Pivekimab, and HT12-GL (sequences provided in Table 13 below), each antibody was conjugated with pHAb dye (Promega, Inc.). The pHAb dye exhibits low fluorescence at neutral extracellular pH but generates strong fluorescence under acidic conditions in endosomes and lysosomes, thereby serving as a direct indicator of cellular internalization. The fluorescence dye-to-antibody ratios (FAR) of CD123.001, Talacotuzumab, Pivekimab, and HT12-GL were 3.26, 3.47, 3.32, and 3.82 respectively. MV4-11 cells were harvested and washed with incubation buffer (IMDM supplemented with 10% FBS). The cells were adjusted to approximately 1.0 x 105cells and incubated with serially diluted pHAb-labeled antibodies (starting concentration: 200 nM, 4-fold serial dilution, 8 concentration points) at 37 °C for 24 hours. Following incubation, cells were washed twice with binding buffer (2% FBS / PBS) and resuspended in cold binding buffer. Fluorescence signals were quantified using flow cytometry. The dose-response plots (FIG. 21) represent mean fluorescence intensity (MFI) as a function of antibody concentration. CD 123.001 exhibited a markedly stronger dose-dependent internalization profile, with significantly higher fluorescence intensity across concentrations compared to Talacotuzumab, Pivekimab, and HT12-GL. As baseline control, cells incubated at 4 °C for 24 hours with 200 nM antibody showed minimal signal (MFI: CD123.001, 693; Talacotuzumab, 660; Pivekimab, 692; HT12-GL, 690.5). These data demonstrate that CD123.001 has an unexpectedly improved ability to internalize.Table 13: CD123 Antibody SequencesExample 18: Target Binding of CD 123 Antibody Conjugates

[0654] To check the target binding of CD123 antibody conjugates, the binding activity was analyzed by ELISA method. The Immuno Clear Standard Module Plates were coated overnight at 4 °C with 100 pL of 2 pg / mL of recombinant human CD 123 protein and blocked with a blocking solution (3% skim milk in PBS, pH 7.4) for 2 h at room temperature. After washing 4-times with washing solution (0.05% Tween-20 in PBS, pH 7.4), several concentrations (4-fold dilution 8 points at 10 pg / mL) of anti-CD123 conjugates in PBS, pH 7.4 were incubated for 1 h at room temperature. After washing 4-times with a washing solution, bound conjugate was detected by HRP-conjugated goat anti-human IgG (H+L) antibody and subsequent incubation with 100 pL of ultra-TMB (3,30,5,50-tetramethylbenzidine) solution for 10 min at room temperature. Then, 100 pL of stop reaction solution (160 mM H2SO4) was added. Absorbance was read at 450 nm on a SpectraMax M5e Multi-Mode plate reader. The binding curve was fitted by 4-parameters sigmoidal in non-linear regression equation among XY analyses using Prism 9.0.2. The results, shown in FIG. 22 and Table 14, indicate that the target binding of the conjugates was similar with various linker-payloads.Table 14: Binding of CD123 ConjugatesExample 19: In Vitro Cytotoxicity of Humanized 32703 Conjugates

[0655] An in vitro cytotoxicity assay was performed using Conjugate C with standard of care (venetoclax and Mylotarg). Test articles were Conjugate C, CD123.001 (which is naked antibody of Conjugate C), GSPT1 Neodegrader (XI), CC-885, venetoclax, and Mylotarg for AML cell lines. Their cytotoxicity was evaluated by using 9 AML cell lines. All these cell lines, except HL60, are known to express CD 123. Cell lines were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of test articles for 4 days. The cell viability was determined by a CTG assay that is luminescence assay (Promega, CellTiter Gio, G7572). Luminescence was read using SpectraMax M5e plate reader. The normalized percentage values were calculated by dividing each treated sample value by the untreated cells, and IC50 values were calculated using the Prism software. As shown in the FIG. 23 it was observed that Conjugate C had cytotoxic activity in CD123 expressing AML cell lines, but not in HL60, and it was more potent than venetoclax and exhibited cytotoxicity comparable to Mylotarg.Example 20: In Vitro Cytotoxicity of Humanized 32703 Conjugates in HL andB-ALL

[0656] To assess the efficacy of Conjugate C in other cell lines, in vitro cytotoxicity test for HL and B-ALL cell lines were performed. Specifically, HDML2, TOM-1, and Nalm-6 cells were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of Conjugate C for 5 days. The cell viability was determined by a Cell Counting Kit-8 (CCK-8) assay that is colorimetric assay (Dojindo Molecular Technologies, Inc). The absorbance at 450 nm was read using the SpectraMax M5e plate reader. The normalized percentage values were calculated by dividing each treated sample value by the untreated cells, and IC50 values were calculated using the Prism software. As shown in FIG. 24 and Table 14, Conjugate C showed potent in vitro cytotoxic for all three of these cell lines. This result demonstrates that Conjugate C exhibits potent efficacy, not only in AML, but also in various hematological malignancies.Table 15: In Vitro Cytotoxicity of Conjugate C in HL and B-ALLExample 21 : Cytotoxicity in TP 53 Isogenic Model

[0657] As demonstrsted herein, Conjugate C exhibits potent in vitro efficacy regardless of TP53 mutation status. The efficacy for TP53 mutations represents an area of significant unmet need in AML, as many patients with TP53-mutated AML do not respond to the current standard of care, such as Venetoclax / azacitidine combo. To confirm the potency of Conjugate C according to TP53 mutation status, another in vitro cytotoxicity assay was designed using a commercially available TP53 KO model (Creative Biogen, CSC-RT2760, which was derived from Molm-13 AML cell line). Molm-13 WT or KO TP53 cells were harvested and resuspended into complete media. Cells were plated into clear flat-bottom 96-well plates. The conjugates, CC885, and venetoclax were diluted into complete media using 3 -fold dilution series. Cells were incubated at 37°C in a humidified 5% CO2 incubator for 3 days. The viability of cells was determined by luminescent CTG assay (Promega, CellTiter Gio, G7572). Luminescence was read using SpectraMax M5e platereader. As shown the FIG. 25, the in vitro cytotoxicity of Conjugate C is not changed regardless of TP53 status. The other GSPT1 degrader, CC-885, also exhibited similar activity between two model. However, the in vitro cytotoxicity of venetoclax decreased by about tenfold in the TP53 knockout model (Table 16). This data demonstrate that Conjugate C exhibits potent in vitro cytotoxicity regardless TP53 status.Table 16: Cytotoxicity (IC50 (M)) in Isogenic TP53 ModelExample 22: In Vitro Cytotoxicity of CD 123 Conjugates with Various Linker-Payloads

[0658] An in vitro cytotoxicity assay was performed to assess the activity of various CD 123 conjugates. The test articles were generated by conjugating CD123.001 with 18 different linkerpayloads, and their cytotoxicity was evaluated using the Nalm-6, clone G5 cell line (FIG. 26). Cells were harvested and seeded into 96-well plates. After incubating overnight, the cells were treated with serial dilutions of conjugates for 72 hrs. Cell viability was measured using the CellTiter- Glo® reagent (Promega), and luminescence was read with a GloMax plate reader (Promega). The luminescent values were normalized for each cell line, and ICso values were calculated using Prism software.

[0659] As shown in FIG. 26 and Table 17, most conjugates exhibited cytotoxic activity against Nalm-6, clone G5 cell line.Table 17: In Vitro Cytotoxicity of CD123 ConjugatesExample 23: In Vitro Evaluation of Conjugate C on Human Myeloid and Erythroid Progenitors using a Colony Forming Cell (CFC) Assay

[0660] The toxicity of conjugate C for human hematological progenitor cells from a healthy donor was assessed in a colony formation assay.

[0661] This assay was set up using pre-qualified human bone marrow cells. Each test article was incubated with human bone marrow cells at serially diluted concentrations. Following 72 hours of incubation, a methylcellulose-based medium was added to each well. Once the cells were added, the tubes were vortexed to ensure equal distribution of the test article throughout the media matrix. A minimum of three replicates was performed for each concentration of the test articles. The replicate dishes were placed at 37 °C in 5% CO2 for 14-16 days. Myeloid and erythroid colonies were evaluated microscopically in situ. Statistical analyses were conducted to assess change in colony numbers. Using this assay, conjugate C demonstrated lower toxicity to human hematopoietic progenitors compared with Mylotarg as shown in FIG 27 A and 27B.

[0662] Example 24: In Vitro Cytotoxicity of CD 123 Conjugates with Various Linker-Payloads

[0663] A dosing analysis was conduted to determine the minimal efficacious dose of Conjugate C. Using the same MV4-11 luciferase disseminated xenograft model as above, doses of 0.03 mg / kg, 0.1 mg / kg, and 1.0 mg / kg were tested. These were each dosed one time intravenously into the tail vein. Efficacy was benchmarked to the standard of care for AML treatment, which is Azacytidine (Aza) combined with Venetoclax (Ven) (50 mg / kg Ven (PO qd x 5 days on / 2 days off x 3 weeks) and 1 mg / kg Aza (PO qd x 5 days)). As shown in FIG. 28A and FIG. 28B, by quantification, Conjugate C at 0.03 mg / kg, did not have much effect vs. vehicle control, butConjugate C at 0.1 mg / kg and 1.0 mg / kg demonstrated substantial reduction in AML tumor burden and survival versus vehicle control. Both tumor burden reduction and survival at these two doses of Conjugate C were superior to the Aza / Ven standard of care treatment. All doses of Conjugate C and Aza / Ven were well tolerated in the animals according to stable body weights recorded in FIG. 29.* * *

[0664] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0665] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0666] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0667] The breadth and scope of the present disclosure should not be limited by any of the abovedescribed exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. An antibody or antigen-binding fragment thereof that immunospecifically binds to CD 123 and comprises a variable heavy chain (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:3, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:8, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NOV, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:7.

2. The antibody or antigen-binding fragment of claim 1, comprising (a) a VH with an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:22; (b) a VH with an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:22; (c) a VH with an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:22; (d) a VH with an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:22; (e) a VH with an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:22; or (f) a VH with an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:21 and / or a VL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:22.

3. The antibody or antigen-binding fragment of claim 2, wherein the VL comprises an amino acid that is not Y position 36 according to Kabat numbering, optionally wherein the VL comprises an F position 36 according to Kabat numbering.

4. The antibody or antigen-binding fragment of any one of claims 1 to 3, comprising a VH comprising the amino acid sequence of SEQ ID NO:21 and / or a VL comprising the amino acid sequence of SEQ ID NO:22.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is an IgG antibody or an antigen-binding fragment thereof, optionally an IgGl antibody or antigen-binding fragment thereof.

6. The antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises (i) an N297A mutation according to EU numbering and / or (ii) a LALA (L234A and L235A) mutation according to EU numbering.

7. The antibody or antigen-binding fragment of claim 6, wherein the antibody or antigenbinding fragment thereof comprises (i) an N297A mutation according to EU numbering.

8. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising the amino acid sequence of any one of SEQ ID NOs:47-53, optionally wherein the constant region comprises the amino acid sequence of SEQ ID NO:51.

9. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a constant region comprising an engineered cysteine.

10. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment comprises (i) a heavy chain comprising the amino acid sequences of SEQ ID NO:55 and / or (ii) a light chain comprising the amino acid sequence of SEQ ID NO:56.

11. A conjugate comprising the antibody or antigen-binding fragment of any one of 1 to 10 conjugated to a GSPT1 neodegrader.

12. A conjugate of formula (I) or formula (II):(II), or a pharmaceutically acceptable salt thereof, wherein: a is 1 to 10; n is 0 or 1;A is phenyl or a C4-Ciocycloalkyl ring;U is selected from NH, O, S, and CF2;R1is independently selected from hydrogen and halo;R10is selected from -CH3, -C(O)R30, -N(R40)2, -(CH2)n OH, -(CH2)nN(R40)2,-(CH2)n Q(CH2)m OH, -(CH2)n Q(CH2)m SH, and -(CH2)n’Q(CH2)mN(R40)2; wherein:R30is hydrogen or Ci-Cealkyl; each R40is independently hydrogen or Ci-Cealkyl;Q is O, S, or NR40; n’ is 1-6; and m’ is 2-5;R20is selected from hydrogen, -(CH2CH2O)v -CH3, C2-Cealkenyl, Ci-Cealkyl; C2- Cealkynyl, benzyl, Cs-Cecycloalkyl, and C3-C6cycloalkyl(Ci-C3alkyl), wherein v’ is from 1 to 24;R50and R51are independently selected from hydrogen and deuterium;X is selected from -NR200-, =C(CH3)-, -Q’-(CH2)n”-, and -Q’(CH2)m”Q”(CH2)n”-; wherein:Q’ and Q” are each independently O, S, or N(R200)v, wherein: v is 1 or 2; each R200is independently hydrogen or Ci-Cealkyl; n” is an integer from 1 to 6; and m’ ’ is an integer from 2 to 6; wherein the left side of each group is attached to L and the right side is attached to A; provided that when X is NH or -Q’-(CH2)n R1is halo; each Y is independently S or O;L is a cleavable linker or non-cleavable linker;L50is a cleavable linker; andBm is the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

13. The conjugate of claim 12, wherein R50and R51are each hydrogen.

14. The conjugate of claim 12 or 13, or a pharmaceutically acceptable salt thereof, wherein a is from 2 to 8.

15. The conjugate of any one of claims 12 to 14, which is a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker.

16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting ofwherein: p is an integer from 1 to 10;is the point of attachment to X; andis the point of attachment to the binding moiety.

17. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting ofwherein: p is an integer from 1 to 10;is the point of attachment to X; andis the point of attachment to the binding moiety.

18. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein L is19. The conjugate of claim 18, or a pharmaceutically acceptable salt thereof, wherein p is 5.

20. The conjugate of any one of claims 12 to 14, which is a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker.

21. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is cleavable by a protease.

22. The conjugate of claim 20 or 21, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting ofwherein: q is an integer from 2 to 10;Z1, Z2, Z3, and Z4are each independently absent or a naturally-occurring amino acid residue in the L- or D-configuration, provided that at least two of Z1, Z2, Z3, and Z4are amino acid residues;the point of attachment to X; andis the point of attachment to the binding moiety.

23. The conjugate of claim 22, or a pharmaceutically acceptable salt thereof, wherein Z1, Z2, Z3, and Z4are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D- glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D- phenylalanine, L-lysine, D-lysine, and glycine; provided that at least two of Z1, Z2, Z3, and Z4are amino acid residues.

24. The conjugate of claim 22 or 23, or a pharmaceutically acceptable salt thereof, wherein:Z1is absent or glycine;Z2is absent or selected from the group consisting of L-glutamine, D-glutamine, L- glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine;Z3is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; andZ4is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D- citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D- phenylalanine, and glycine.

25. The conjugate of any one of claims 22 to 24, or a pharmaceutically acceptable salt thereof, wherein L is26. The conjugate of claim 14, or a pharmaceutically acceptable salt thereof, wherein q is 5.

27. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein L is a bioreducible linker.

28. The conjugate of claim 27, wherein L is selected from the group consisting ofq is an integer from 2 to 10;R, R’, R”, and R’” are each independently selected from hydrogen, Ci- CealkoxyCi-Cealkyl, (Ci-Ce iNCi-Cealkyl, and Ci-Cealkyl, or, two geminal R groups, together with the carbon atom to which they are attached, can form a cyclobutyl or cyclopropyl ring;the point of attachment to X; and*is the point of attachment to the binding moiety.

29. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein L is an acid cleavable linker.

30. The conjugate of claim 29, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting ofwherein: q is an integer from 2 to 10;the point of attachment to X; and*is the point of attachment to the binding moiety.

31. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein L is a click-to-release linker.

32. The conjugate of claim 31, or a pharmaceutically acceptable salt thereof, wherein L is selected fromwherein: q is an integer from 2 to 10;is the point of attachment to X; andis the point of attachment to the binding moiety.

33. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein L is a pyrophosphatase cleavable linker.

34. The conjugate of claim 33, or a pharmaceutically acceptable salt thereof, wherein L iswherein: q is an integer from 2 to 10;is the point of attachment to X; andis the point of attachment to the binding moiety.

35. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein L is a beta-glucuronidase cleavable linker.

36. The conjugate of claim 35, or a pharmaceutically acceptable salt thereof, wherein L is selected fromq is an integer from 2 to 10;— is absent or a bond;the point of attachment to X; and *is the point of attachment to the binding moiety.

37. The conjugate of claim 36, or a pharmaceutically acceptable salt thereof, wherein L is38. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is halo; andX is -N(R200)v(CH2)m”O(CH2)n”-; wherein: v is 1; m’ ’ and n’ ’ are 2; and R200is methyl.

39. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is halo; andX is -N(R200)v(CH2)m”O(CH2)n”-; wherein: v is 2; m’ ’ and n’ ’ are 2; and each R200is methyl.

40. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is halo; andX is -O(CH2)n”-; wherein: n” is 2.

41. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is halo; andX is -S(CH2)n”-; wherein: n” is 2.

42. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is hydrogen; andX is -NR200-; wherein:R200is methyl.

43. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is halo; andX is -NR200-; wherein:R200is hydrogen.

44. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is halo; andX is -NR2-; wherein:R2is hydrogen.

45. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is phenyl;U is NH;R1is hydrogen; andX is -C(CH3)=.

46. The conjugate of any one of claims 12 to 37, which is a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein:A is a C4-Ciocycloalkyl ring;U is NH;R1is hydrogen; andX is -N(R200)(CH2)mO(CH2)n-; wherein: n” is 1; m” is 2; andR200is methyl.

47. The conjugate of any one of claims 12 to 14 which is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is cleavable by a protease.

48. The conjugate of claim 47, wherein L50is selected from the group consisting ofwherein: q is from 2 to 10;Z1, Z2, Z3, Z4, and Z5are each independently absent or a naturally-occurring amino acid residue in the L- or D-configuration, provided that at least two of Z1, Z2, Z3, Z4, and Z5are amino acid residues;is the point of attachment to the parent molecular moiety; andis the point of attachment to the binding moiety.

49. The conjugate of claim 48, or a pharmaceutically acceptable salt thereof, wherein Z1, Z2, Z3, Z4, and Z5are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D- glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D- phenylalanine, L-lysine, D-lysine, and glycine; provided that at least two of Z1, Z2, Z3, Z4, and Z5are amino acid residues.

50. The conjugate of claim 48 or 49, or a pharmaceutically acceptable salt thereof, wherein:Z1is absent or glycine;Z2is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine;Z3is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L- phenylalanine, D-phenylalanine, and glycine;Z4is selected from the group consisting of L-citrulline, D-citrulline, L-asparagine, D- asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine; andZ5is absent or glycine.

51. The conjugate of any one of claim 48 to 50, or a pharmaceutically acceptable salt thereof, wherein L50is52. The conjugate of claim 51, wherein q is 4.

53. The conjugate of any one of claims 12 to 14 which is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein L50is a bioreducible linker.

54. The conjugate of claim 53, or a pharmaceutically acceptable salt thereof, wherein L50iswherein:q is from 2 to 10;R, R’, R”, and R’” are each independently selected from hydrogen, Ci-CealkoxyCi- Cealkyl, (Ci-Cealkyl^NCi-Cealkyl, and Ci-Cealkyl, or, two geminal R groups, together with the carbon atom to which they are attached, can form a cyclobutyl or cyclopropyl ring;is the point of attachment to the parent molecular moiety; andis the point of attachment to the binding moiety.

55. The conjugate of claim 53 or 54, or pharmaceutically acceptable salt thereof, wherein L50is56. The conjugate of claim 55, or a pharmaceutically acceptable salt thereof, wherein q is 2.

57. The conjugate of any one of claims 12 to 14 which is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein L50is a click-to-release linker.

58. The conjugate of claim 57, or a pharmaceutically acceptable salt thereof, wherein L50iswherein: q is from 2 to 10;is the point of attachment to the parent molecular moiety; andis the point of attachment to the binding moiety.

59. The conjugate of any one of claims 12 to 14 which is a conjugate of formula (II), or a pharmaceutically acceptable salt thereof, wherein L50is a beta-glucuronidase cleavable linker.

60. The conjugate of claim 59, or a pharmaceutically acceptable salt thereof, wherein L50is selected fromq is from 2 to 10;— is absent or a bond;is the point of attachment to the parent molecular moiety; andis the point of attachment to the binding moiety.

61. The conjugate of any one of claims 12 to 60, or a pharmaceutically acceptable salt thereof, wherein a is from 3 to 4.

62. The conjugate of any one of claims 12 to 60, or a pharmaceutically acceptable salt thereof, wherein a is 8.

63. The conjugate of any one of claims 12 to 62, wherein the L or L50is attached to a cysteine in the binding moiety, optionally wherein the conjugation is irreversible.wherein Bm is antibody CD123.001 and a is 2-8, optionally 3-5.

65. A composition comprising at least one conjugate of any one of claims 11 to 64, wherein the average number of neodegraders per Bm is about 2 to about 8, optionally wherein the average number of neodegraders per Bm is about 3 to about 5 or wherein the average number of neodegraders per Bm is about 3.5 to 4.5.

66. A method of treating a hematological malignancy in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the conjugate of any one of claims 11 to 64 or a pharmaceutically acceptable salt thereof, or the composition of claim 65.

67. The method of claim 66, wherein the hematological malignancy is CD 123 -positive hematological malignancy.

68. The method of claim 66 or 67, wherein the hematological malignancy is a leukemia or a lymphoma.

69. The method of any one of claims 66-68, wherein the hematological malignancy is an acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, Hodgkin lymphoma, or blastic plasmacytoid dendritic neoplasm (BPDCN).

70. The method of claim 66 or 67, wherein the hematological malignancy is present as minimal residual disease (MRD).

71. The method of any one of claims 66 to 70, wherein the hematological malignancy is AML, optionally wherein the AML is disseminated AML.

72. The method of any one of claims 66 to 71, wherein the subject is an unfit subject.

73. The method of any one of claims 66 to 72, wherein the hematological malignancy has a TP 53 mutation.

74. The method of any one of claims 66 to 73, wherein the hematological malignancy has a FLT3 mutation, optionally wherein the in the mutation is an internal tandem duplication (ITD).

75. The method of any one of claims 66 to 74, wherein the subject has previously been treated with venetoclax and hypomethylating agent (HMA).

76. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the conjugate of any one of claims 11 to 64 or a pharmaceutically acceptable salt thereof or the composition of claim 65 in the preparation of a medicament for use in the method of any one of claims 66 to 75.

77. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the conjugate of any one of claims 11 to 64 or a pharmaceutically acceptable salt thereof, or composition of claim 64 for use in the method of any one of claims 66 to 75.

78. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding fragment thereof of any one of claims 1-10.

79. An isolated polynucleotide comprising a nucleic acid molecule encoding the light chain variable region or light chain of the antibody or antigen-binding fragment thereof of any one of claims 1-10.

80. An isolated polynucleotide comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-10.

81. An isolated vector comprising the polynucleotide of any one of claims 78-80.

82. A host cell comprising (a) the polynucleotide of any one of claims 78-80, (b) the vector of claim 81, or (c) a first vector comprising the polynucleotide of claim 78 and a second vector comprising the polynucleotide of claim 79.

83. The host cell of claim 82, wherein the host cell is a CHO cell or a CHO-K1 cell.

84. A method of producing an antibody or antigen-binding fragment thereof that immunospecifically binds to CD123 comprising culturing the host cell of claim 82 or 83 so that the nucleic acid molecule is expressed and the antibody or antigen-binding fragment thereof is produced, optionally wherein the method further comprises isolating the antibody or antigenbinding fragment thereof from the culture.

85. An antibody or antigen-binding fragment thereof that immunospecifically binds to CD123 and is encoded by the polynucleotide of any one of claims 74-80 or produced by the method of claim 84.

86. A method of making a conjugate comprising conjugating the antibody or antigen-binding fragment thereof of any one of claims 1-10 to a GSPT1 degrader.

87. A conjugate produced by the method of claim 86.

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