TW202434289A - Antibody-drug conjugates (a2dcs) with enzymatically cleavable groups - Google Patents

Abstract

This application relates to antibody-2-drug conjugates (A2DCs) which comprise a plurality of kinesin spindle protein inhibitor payloads, optionally having different permeability profiles, which may have advantageous payload release properties within a tumor microenvironment, pharmaceutical compositions, processes for preparation thereof, and the use thereof for treating, preventing, or managing diseases and conditions including hyperproliferative disorders, such as, cancer in humans and other mammals.

Description

Antibody-drug conjugate with enzyme cleavable group (A2DCS)

This application relates to antibody-drug conjugates, pharmaceutical compositions, methods of preparing the same, and their use for treating, preventing or controlling diseases and conditions, including hyperproliferative disorders (such as cancer), in humans and other mammals.

In one aspect, provided herein is an antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof: (I) wherein: AK ¹ is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via a sulfur atom of cysteine, an antibody linked via a nitrogen atom of lysine, or an antibody linked via both a sulfur atom of cysteine and a nitrogen atom of lysine); X ¹ and X ² are each independently -NH- or -S-; L ¹ and L ² are each independently a stabilizing linker; ^RA is a first protease-cleavable spinning hammer kinesin inhibitor group; ^RB is a second protease-cleavable spinning hammer kinesin inhibitor group; wherein ^RA and ^RB are different groups; and a and b are each independently an integer from 1 to 50.

In another aspect, provided herein is an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof: (II) wherein: AK ² is an antibody linked via a sulfur atom or via a nitrogen atom; L ³ is a stabilizing linker; ^RC and ^RD are each independently a protease-cleavable spinodal kinesin inhibitor group; and z is an integer from 1 to 50.

In another aspect, provided herein is an antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt thereof: (III) wherein: AK ³ is an antibody or an antigen-binding fragment thereof linked via a sulfur atom or via a nitrogen atom; L ⁴ is a stabilizing linker; KSP ⁵ is a first spindle-dried kinesin inhibitor; P ⁵ is a protease-cleavable linker; KSP ⁶ is a second spindle-dried kinesin inhibitor; and y is an integer from 1 to 50.

In some embodiments, provided herein is a pharmaceutical composition comprising an antibody-drug conjugate described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically suitable inert non-toxic auxiliary agent.

In some embodiments, provided herein is a method for treating or preventing a disease, the method comprising administering to a patient in need thereof an antibody-drug conjugate described herein.

In some embodiments, provided herein is a method of treating or preventing a hyperproliferative or angiogenic disorder comprising administering to a patient in need thereof an antibody-drug conjugate described herein.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that a publication, patent, or patent application incorporated by reference conflicts with the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such conflicting material. Cross-References

This application claims the benefit of European Patent Application No. EP22306697.8 filed on November 17, 2022, which is incorporated herein by reference in its entirety.

This application contains a sequence listing, which is submitted herewith in XML format and is hereby incorporated by reference in its entirety. The XML copy created on November 15, 2023 is named 59362-724.601 SL.xml and is 151 kilobytes in size. Background

Antibody-drug conjugates (ADCs) are an emerging class of drugs that utilize antibodies to improve cytotoxicity or targeting of immuno-oncology payloads to tumor cells. In most cases, active metabolites are formed upon internalization of the ADC into cancer cells, where they are either cleaved by enzymes of the linker or degraded by the antibody. The approval of 12 ADCs at the same time demonstrates the potential of ADCs to improve cancer therapy.

The drug-to-antibody ratio (DAR) characterizes the number of payloads attached to the antibody. A higher DAR generally results in more efficient payload delivery and therefore can result in higher ADC potency. However, due to the high hydrophobicity of most payload classes, an increase in the DAR generally results in poor physicochemical properties of the ADC. The result is an increased sensitivity of the ADC to aggregate formation, higher nonspecific absorption levels in non-tumor cells leading to stronger side effects, and higher clearance of the ADC and poor PK parameters.

One approach to overcome these tendencies is to employ highly hydrophilic and multivalent polymers to produce ADCs with high drug loading without compromising physicochemical and pharmacokinetic properties (e.g. A. V. Yurkovetskiy in Cancer Research (2015), 75(16), 3365-3372), but this adds another layer of complexity to the ADC.

Another critical aspect of ADC efficacy, which depends on the tumor to be treated, is the physicochemical profile of the released payload.

The release of cell-permeable payloads can be associated with bystander killing effects, which are considered to be particularly beneficial for treating tumors with heterogeneous target expression (J. A. Costoplus, ACS Med Chem Lett, 2019, 10, 1393). On the other hand, this cell-permeable metabolite profile can increase side effects due to redistribution of the released payload or premature payload cleavage, both of which can cause damage to healthy cells.

In contrast, the release of an impermeable payload can lead to intracellular retention and accumulation of the active drug inside tumor cells, which can have a beneficial effect on potency. At the same time, this profile can increase the safety of the ADC, since the released impermeable payload will not induce healthy cell damage. However, such metabolite designs cannot achieve any bystander killing of the released payload. Therefore, it may be beneficial to combine the two metabolite profiles released by tumor-related proteases to achieve optimal ADC efficacy with a good safety profile.

KSP (synonyms: Eg5, KIF11) is an ATP-dependent motor protein involved in centrosome separation during the G2/M phase of the cell cycle (S. M. Myers et al., Fut. Med. Chem. 2016, 8, 463). Blocking this essential event in mitosis with small molecule KSP inhibitors (KSPis) resulted in high anti-tumor efficacy, attracting great attention to this class of compounds (H. B. El-Nassan, Eur. J. Med. Chem. 2013, 62, 614). However, translating the preclinical efficacy of SMOL KSP inhibitors into highly effective clinical regimens with an acceptable therapeutic range remains challenging. Only proliferating cells in the G2M phase of the cell cycle are sensitive to small molecule KSP inhibitors (SMOL KSPi). Therefore, to be effective, SMOL KSPi may require frequent dosing, which often leads to dose-limiting side effects such as neutropenia.

Recently, inhibitors of kinesin (KSP) have been described as a novel and highly effective class of payloads in ADCs (H.-G. Lerchen, Angew. Chem. Int. Ed. 2018, 57, 15243; WO2015/096982). These KSPi payload molecules offer a variety of options for payload linkers that are compatible with cleavable or non-cleavable linking chemistries.

Legumain is an asparaginyl peptidyl endonuclease that is overexpressed in solid tumors and is associated with invasion, metastasis, and poor survival (Dall, E. and Brandstetter, H. (2016) Biochimie 2016, 22, 126−50).

In WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578, it is claimed that different ADCs are protected which are activated by the tumor-associated protease tadalafil to release the active KSPi payload. The profile of the cleaved KSPi payload can be modified and tuned for different physicochemical properties to achieve different degrees of membrane permeability without destroying the inhibition of the KSP target. Detailed description of the invention

The profile of the SMOL KSPi described above and those described as ADC payloads in patent applications WO2015/096982, WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578, among others, allows for the novel design of highly potent antibody-2-drug conjugates (A2DCs).

The antibody-2-drug conjugates of the present invention are described by formula (I), formula (I'), formula (II), formula (II') and formula (III): (I) (I') (II) (II') (III) wherein: each KSP (e.g., KSP ¹ , KSP ² , KSP ³ , KSP ⁴ , KSP ⁵ , and KSP ⁶ ) is a spinosome-derived kinesin inhibitor; each R (e.g., ^RA , ^RB , ^RC , and ^RD ) is independently a protease-cleavable spinosome-derived kinesin inhibitor group; each P (e.g., P ¹ , P ² , P 3, P ⁴ , and P ⁵ ) is a protease-cleavable linker; each L (e.g., L ¹ , L ² , L ³ , and L ⁴ ) is a non-cleavable linker; each AK (e.g., AK ¹ , AK ² , and AK ³ ) is an antibody or an antigen-binding fragment thereof, wherein the antibody may be a monoclonal antibody or a fragment ^thereof ; X ¹ and X ² are each independently nitrogen or sulfur; and each of a, b, y and z is an integer between 1 and 50.

In some embodiments, the compound is an antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof: (I) wherein: AK ¹ is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via a sulfur atom of cysteine, an antibody linked via a nitrogen atom of lysine, or an antibody linked via both a sulfur atom of cysteine and a nitrogen atom of lysine); X ¹ and X ² are each independently -NH- or -S-; L ¹ and L ² are each independently a stabilizing linker; ^RA is a first protease-cleavable spinning hammer kinesin inhibitor group; ^RB is a second protease-cleavable spinning hammer kinesin inhibitor group; wherein ^RA and ^RB are different groups; and a and b are each independently an integer from 1 to 50.

In some embodiments, ^RA is a first protease-cleavable spinning hammer protein inhibitor of formula (i): (i) KSPi ¹ -P ¹ - ^# ; wherein KSPi ¹ is a first spinning hammer protein inhibitor, P ¹ is a first protease-cleavable linker; and ^# is a bond between ^RA and L ¹ ; and ^RB is a second protease-cleavable spinning hammer protein inhibitor of formula (ii): (ii) ^## -P ² -KSPi ² ; wherein ^## is a bond between L ² and ^RB ; P ² is a second protease-cleavable linker; KSPi ² is a second spinning hammer protein inhibitor, and wherein ^RA and ^RB are different groups.

In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof has the structure of formula (I'): (I') wherein: AK ¹ is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via the sulfur atom of cysteine, an antibody linked via the nitrogen atom of lysine, or an antibody linked via both the sulfur atom of cysteine and the nitrogen atom of lysine); X ¹ and X ² are each independently -NH- or -S-; L ¹ and L ² are each independently: , , , , , , , , or , ^P1 is a first protease-cleavable linker; ^P2 is a second protease-cleavable linker; KSPi ¹ is: ; KSPi ² is: ; R ¹ and R ⁶ are each independently a halogen; R ² and R ⁷ are each independently a C _1-6 alkyl; m and n are each independently an integer from 1 to 5; and a and b are each independently an integer from 1 to 50.

In some embodiments, ^P1 is a first protease cleavable linker. In some embodiments, ^P2 is a second protease cleavable linker. In some embodiments, ^P1 and ^P2 are each independently a histone protease or a soy protein cleavable linker. In some embodiments, ^P1 and ^P2 are each independently a soy protein cleavable linker. In some embodiments, ^P1 and ^P2 are each independently a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.

In some embodiments, the antibody-drug conjugate is a compound of formula (I), wherein: AK ¹ is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via the sulfur atom of cysteine, an antibody linked via the nitrogen atom of lysine, or an antibody linked via both the sulfur atom of cysteine and the nitrogen atom of lysine); X ¹ and X ² are each independently -NH- or -S-; L ¹ and L ² are each independently: , , , , , , , , or ; ^RA is: ; ^RB is: ; wherein: R ¹ and R ⁶ are each independently a halogen; R ² and R ⁷ are each independently a C _1-6 alkyl; R ³ and R ⁸ are each independently a C _1-6 alkyl or -C _1-6 alkylene-COOH; R ⁴ , R ⁵ , R ⁹ and R ¹⁰ are each independently hydrogen or C _1-6 alkyl; a and b are each independently an integer from 1 to 50; m and n are each independently an integer from 1 to 5; and p, q, r and s are each independently 0, 1, 2, 3 or 4.

In some embodiments, AK ¹ is anti-EGFR, anti-Her2, anti-TWEAKR, anti-CD123 or anti-CXCR5. In some embodiments, AK ¹ is anti-EGFR mAb, anti-Her2 mAb, anti-TWEAKR mAb, anti-CD123 mAb or anti-CXCR5 mAb.

In some embodiments, ^X1 and ^X2 are each -NH-. In some embodiments, ^X1 and ^X2 are each -S-. In some embodiments, ^X1 is -NH- and ^X2 is -S-. In some embodiments, ^X1 is -S- and ^X2 is -NH-.

In some embodiments, ^L1 and ^L2 are each independently: , , , , , , or .

In some embodiments, ^L1 and ^L2 are each independently: , , , , , or .

In some embodiments, ^L1 and ^L2 are each independently: or .

In some embodiments, each R ¹ is independently halogen. In some embodiments, each R ¹ is chloro, fluoro, or bromo. In some embodiments, each R ¹ is independently chloro or fluoro. In some embodiments, each R ¹ is independently fluoro.

In some embodiments, each R ² is independently C _1-6 alkyl. In some embodiments, each R ² is independently C _3-6 alkyl. In some embodiments, each R ² is independently n-propyl, isopropyl, n-butyl, tert-butyl or isobutyl. In some embodiments, each R ² is independently -C(CH ₃ ) ₃ .

In some embodiments, each R ³ is independently C _1-6 alkyl or -C _1-6 alkylene-COOH. In some embodiments, each R ³ is independently C _1-3 alkyl or -C _1-3 alkylene-COOH. In some embodiments, each R ³ is independently methyl, ethyl, propyl or -C _1-3 alkylene-COOH. In some embodiments, each R ³ is independently methyl. In some embodiments, each R ³ is independently -CH ₂ COOH.

In some embodiments, each R ⁴ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ⁴ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ⁴ is independently hydrogen, methyl or ethyl. In some embodiments, each R ⁴ is independently hydrogen or methyl. In some embodiments, each R ⁴ is hydrogen. In some embodiments, each R ⁴ is methyl.

In some embodiments, each R ⁵ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ⁵ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ⁵ is independently hydrogen, methyl or ethyl. In some embodiments, each R ⁵ is independently hydrogen or methyl. In some embodiments, each R ⁵ is hydrogen. In some embodiments, each R ⁵ is methyl.

In some embodiments, each R ⁶ is independently halogen. In some embodiments, each R ⁶ is chloro, fluoro or bromo. In some embodiments, each R ⁶ is independently chloro or fluoro. In some embodiments, each R ⁶ is independently fluoro.

In some embodiments, each R ⁷ is independently C _1-6 alkyl. In some embodiments, each R ⁷ is independently C _3-6 alkyl. In some embodiments, each R ⁷ is independently n-propyl, isopropyl, n-butyl, tertiary butyl or isobutyl. In some embodiments, each R ⁷ is independently -C(CH ₃ ) ₃ .

In some embodiments, each R ⁸ is independently C _1-6 alkyl or -C _1-6 alkylene-COOH. In some embodiments, each R ⁸ is independently C _1-3 alkyl or -C _1-3 alkylene-COOH. In some embodiments, each R ⁸ is independently methyl, ethyl, propyl or -C _1-3 alkylene-COOH. In some embodiments, each R ⁸ is independently methyl. In some embodiments, each R ⁸ is independently -CH ₂ COOH.

In some embodiments, each R ⁹ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ⁹ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ^{9 is independently hydrogen, methyl or ethyl. In some embodiments, each R 9 is} independently hydrogen or methyl. In some embodiments, each R ⁹ is hydrogen. In some embodiments, each R ⁹ is methyl.

In some embodiments, each R ¹⁰ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ¹⁰ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ¹⁰ is independently hydrogen, methyl or ethyl. In some embodiments, each R ¹⁰ is independently hydrogen or methyl. In some embodiments, each R ¹⁰ is hydrogen. In some embodiments, each R ¹⁰ is methyl.

In some embodiments, a and b are each independently an integer from 1 to 50. In some embodiments, a is an integer from 1 to 25. In some embodiments, a is an integer from 1 to 20. In some embodiments, a is an integer from 1 to 15. In some embodiments, a is an integer from 1 to 10. In some embodiments, a is an integer from 1 to 5. In some embodiments, b is an integer from 1 to 25. In some embodiments, b is an integer from 1 to 20. In some embodiments, b is an integer from 1 to 15. In some embodiments, b is an integer from 1 to 10. In some embodiments, b is an integer from 1 to 5.

In some embodiments, m is an integer from 1 to 5. In some embodiments, m is an integer from 1 to 4. In some embodiments, m is an integer from 1 to 3. In some embodiments, m is 1 or 2. In some embodiments, m is 2. In some embodiments, m is 1.

In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments, n is 2. In some embodiments, n is 1.

In some embodiments, p is 0, 1, 2, 3, or 4. In some embodiments, p is 0, 1, 2, or 3. In some embodiments, p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3.

In some embodiments, q is 0, 1, 2, 3, or 4. In some embodiments, q is 0, 1, 2, or 3. In some embodiments, q is 0, 1, or 2. In some embodiments, q is 0 or 1. In some embodiments, q is 0. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

In some embodiments, r is 0, 1, 2, 3, or 4. In some embodiments, r is 0, 1, 2, or 3. In some embodiments, r is 0, 1, or 2. In some embodiments, r is 0 or 1. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3.

In some embodiments, s is 0, 1, 2, 3, or 4. In some embodiments, s is 0, 1, 2, or 3. In some embodiments, s is 0, 1, or 2. In some embodiments, s is 0 or 1. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3.

In some embodiments, the R ^A -L ¹ -X ¹ -fragment is: or .

In some embodiments, the -X ² -L ^{2 -RB} fragment is: or .

In some embodiments, the antibody-drug conjugate of Formula (I) has the structure of Formula (IA), or a pharmaceutically acceptable salt thereof: (IA).

In some embodiments, the antibody drug conjugate is: , , , , or .

In some embodiments, the antibody-drug conjugate of Formula (I) has the structure of Formula (IB), or a pharmaceutically acceptable salt thereof: (IB).

In some embodiments, the antibody drug conjugate is: or .

The antibody-drug conjugate of formula (I) has the structure of formula (IC), or a pharmaceutically acceptable salt thereof: (IC).

In some embodiments, the antibody drug conjugate is: , , or .

In some embodiments, the compound is an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof: (II) wherein: AK ² is an antibody linked via a sulfur atom or via a nitrogen atom; L ³ is a stabilizing linker; ^RC and ^RD are each independently a protease-cleavable spinodal kinesin inhibitor group; and z is an integer from 1 to 50.

In some embodiments, the antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof has the structure of formula (II'): (II') wherein: AK ² is an antibody linked via the sulfur atom of cysteine, via the nitrogen atom of lysine, or via a combination thereof; L ³ is a stabilizing linker; P ³ and P ⁴ are each independently a protease-cleavable linker; KSPi ³ and KSPi ⁴ are each independently a spinophilic kinesin inhibitor; and z is an integer between 1 and 50.

In some embodiments, KSPi ³ and KSPi ⁴ are each independently: or ; wherein: each R ¹¹ is a halogen; and n is an integer from 1 to 5.

In some embodiments, ^P3 and ^P4 are each independently a protease-cleavable linker. In some embodiments, ^P3 and ^P4 are each independently a histone protease-cleavable linker or a soy protein-cleavable linker. In some embodiments, ^P3 and ^P4 are each independently a soy protein-cleavable linker. In some embodiments, ^P3 and ^P4 are each independently a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.

In some embodiments, the antibody conjugate or a pharmaceutically acceptable salt thereof, wherein: ^RC is ; R ^D is or ; wherein: each R ¹¹ is a halogen; each R ¹² is a C _1-6 alkyl group or -C _1-6 alkylene-CONH ₂ ; each R ¹³ is a C _1-6 alkyl group; each R ¹⁴ is hydrogen or a C _1-6 alkyl group; each R ¹⁵ is hydrogen or a C _1-6 alkyl group; each R ¹⁶ is a C _1-6 alkyl group or -C _1-6 alkylene-COOH; each R ¹⁷ is hydrogen or a C _1-6 alkyl group; n is an integer from 1 to 5; and r, s and t are each independently 0, 1, 2, 3 or 4.

In some embodiments, AK ² is anti-EGFR, anti-Her2, anti-TWEAKR, anti-CD123 or anti-CXCR5. In some embodiments, AK ² is anti-EGFR mAb, anti-Her2 mAb, anti-TWEAKR mAb, anti-CD123 mAb or anti-CXCR5 mAb.

In some embodiments, ^L3 is: , , , , , , , , , , , or .

In some embodiments, each R ¹¹ is independently halogen. In some embodiments, each R ¹¹ is chloro, fluoro, or bromo. In some embodiments, each R ¹¹ is independently chloro or fluoro. In some embodiments, each R ¹¹ is independently fluoro.

In some embodiments, each R ¹² is independently C _1-6 alkyl or -C _1-6 alkylene-COOH. In some embodiments, each R ¹² is independently C _1-3 alkyl or -C _1-3 alkylene-COOH. In some embodiments, each R ¹² is independently methyl, ethyl, propyl or -C _1-3 alkylene-COOH. In some embodiments, each R ¹² is independently methyl. In some embodiments, each R ¹² is independently -CH ₂ COOH.

In some embodiments, each R ¹³ is independently C _1-6 alkyl. In some embodiments, each R ¹³ is independently C _1-3 alkyl. In some embodiments, each R ¹³ is independently methyl, ethyl, n-propyl or isopropyl. In some embodiments, each R ¹³ is independently methyl, n-propyl or isopropyl. In some embodiments, each R ¹³ is methyl. In some embodiments, each R ¹³ is isopropyl.

In some embodiments, each R ¹⁴ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ¹⁴ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ^{14 is independently hydrogen, methyl or ethyl. In some embodiments, each R 14 is} independently hydrogen or methyl. In some embodiments, each R ¹⁴ is hydrogen. In some embodiments, each R ¹⁴ is methyl.

In some embodiments, each R ¹⁵ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ¹⁵ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ¹⁵ is independently hydrogen, methyl, ethyl or propyl. In some embodiments, each R ¹⁵ is independently hydrogen, methyl or ethyl. In some embodiments, each R 15 is independently ^{hydrogen or methyl. In some embodiments, each R 15 is hydrogen. In some embodiments, each R 15 is methyl} .

In some embodiments, each R ¹⁶ is independently C _1-6 alkyl or -C _1-6 alkylene-COOH. In some embodiments, each R ¹⁶ is independently C _1-6 alkyl. In some embodiments, each R ¹⁶ is -C _1-6 alkylene-COOH. In some embodiments, each R ¹⁶ is -C _1-5 alkylene-COOH. In some embodiments, each R ¹⁶ is -C _1-4 alkylene-COOH. In some embodiments, each R ¹⁶ is independently -C _1-3 alkylene-COOH. In some embodiments, each R ¹⁶ is -C _1-2 alkylene-COOH. In some embodiments, each R ¹⁶ is -CH ₂ COOH.

In some embodiments, each R ¹⁷ is independently hydrogen or C _1-6 alkyl. In some embodiments, each R ¹⁷ is independently hydrogen or C _1-3 alkyl. In some embodiments, each R ¹⁷ is independently hydrogen, methyl, ethyl or propyl. In some embodiments, each R ¹⁷ is independently hydrogen, methyl or ethyl. In some embodiments, each R 17 is independently ^{hydrogen or methyl. In some embodiments, each R 17 is hydrogen. In some embodiments, each R 17 is methyl} .

In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments, n is 2. In some embodiments, n is 1.

In some embodiments, r is 0, 1, 2, 3, or 4. In some embodiments, r is 0, 1, 2, or 3. In some embodiments, r is 0, 1, or 2. In some embodiments, r is 0 or 1. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3.

In some embodiments, s is 0, 1, 2, 3, or 4. In some embodiments, s is 0, 1, 2, or 3. In some embodiments, s is 0, 1, or 2. In some embodiments, s is 0 or 1. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3.

In some embodiments, t is 0, 1, 2, 3, or 4. In some embodiments, t is 0, 1, 2, or 3. In some embodiments, t is 0, 1, or 2. In some embodiments, t is 0 or 1. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3.

In some embodiments, ^RC is: , , or .

In some embodiments, ^RD is: , , , , , or .

In some embodiments, the antibody drug conjugate is: , , , , , , , , , , , , , , or .

In some embodiments, the compound is an antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt thereof: (III) wherein: AK ³ is an antibody or an antigen-binding fragment thereof linked via a sulfur atom or via a nitrogen atom; L ⁴ is a stabilizing linker; KSP ⁵ is a first spindle-dried kinesin inhibitor; P ⁵ is a protease-cleavable linker; KSP ⁶ is a second spindle-dried kinesin inhibitor; and y is an integer from 1 to 50.

In some embodiments, L ⁴ is a substituted or unsubstituted C _1-60 alkyl linker, or a substituted or unsubstituted 1-60 membered heteroalkyl linker. In some embodiments, L ⁴ has the following structure: or ; where #AK ³ represents the key between L ⁴ and AK ³ ; and # represents the key between L ⁴ and KSPi ⁵ .

In some embodiments, the antibody-drug conjugate is a compound of formula (III), wherein: KSPi ⁵ is a first spinodal kinesin inhibitor having the following structure: ; KSPi ⁶ is a second spindle-driven kinesin inhibitor having the following structure: , or ; wherein: # represents the bond between L ⁴ and KSPi ⁵ ; ## represents the bond between KSPi ⁵ and P ⁵ ; ### represents the bond between P ⁵ and KSPi ⁶ ; each R ²¹ is independently a halogen; each R ²² is independently a C _1-6 alkyl; and n is an integer from 1 to 5.

In some embodiments, ^P5 is a protease-cleavable linker. In some embodiments, ^P5 is a histone protease-cleavable linker or a soy protein-cleavable linker. In some embodiments, ^P5 is a soy protein-cleavable linker. In some embodiments, ^P5 is a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.

In some embodiments, ^P5 is a soy protein cleavable linker having the following structure: ^-P5a - ^L5 - ^P5b- wherein: ^P5a and ^P5b are each independently a protease cleavable linker; and ^L5 is a stable linker.

In some embodiments, ^P5a and ^P5b are each independently a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.

In some embodiments, L ⁵ is a stable linker, which is a substituted or unsubstituted C _1-60 alkyl linker, or a substituted or unsubstituted 1-60 member heteroalkyl linker. In some embodiments, L 5 is a stable linker, which is a substituted or unsubstituted C _1-50 alkyl linker, or a substituted or unsubstituted 1-50 member heteroalkyl linker. In some embodiments, L ⁵ is a stable linker, which is a substituted or unsubstituted C _1-40 alkyl linker, ^{or a substituted or unsubstituted 1-40 member heteroalkyl linker. In some embodiments, L 5 is} a stable linker, which is a substituted or unsubstituted C _1-30 alkyl linker, or a substituted or unsubstituted 1-30 member heteroalkyl linker. In some embodiments, L ⁵ is a stable linker, which is a substituted or unsubstituted C _1-20 alkyl linker, or a substituted or unsubstituted 1-20 membered heteroalkyl linker. In some embodiments, L ⁵ is a stable linker, which is a substituted or unsubstituted C _1-10 alkyl linker, or a substituted or unsubstituted 1-10 membered heteroalkyl linker.

In some embodiments, ^P5 is a soy protein cleavable linker having the following structure: , , , or .

In some embodiments, AK ³ is anti-EGFR, anti-Her2, anti-TWEAKR, anti-CD123 or anti-CXCR5. In some embodiments, AK ³ is anti-EGFR mAb, anti-Her2 mAb, anti-TWEAKR mAb, anti-CD123 mAb or anti-CXCR5 mAb.

In some embodiments, each R ²¹ is independently halogen. In some embodiments, each R ²¹ is chlorine, fluorine or bromine. In some embodiments, each R ²¹ is independently chlorine or fluorine. In some embodiments, each R ²¹ is independently fluorine.

In some embodiments, each R ²² is independently C _1-6 alkyl. In some embodiments, each R ²² is independently C _1-4 alkyl. In some embodiments, each R ²² is independently n-propyl, isopropyl, n-butyl, isobutyl, dibutyl or tertiary butyl. In some embodiments, each R ²² is independently tertiary butyl.

In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments, n is 2. In some embodiments, n is 1.

In some embodiments, y is an integer from 1 to 50. In some embodiments, y is an integer from 1 to 25. In some embodiments, y is an integer from 1 to 20. In some embodiments, y is an integer from 1 to 15. In some embodiments, y is an integer from 1 to 10. In some embodiments, y is an integer from 1 to 6. In some embodiments, y is an integer from 2 to 6.

In some embodiments, KSPi ⁵ is: ; and KSPi ⁶ is or ; Wherein: # represents the bond between L ⁴ and KSPi ⁵ ; ## represents the bond between KSPi ⁵ and P ⁵ ; and ### represents the bond between P ⁵ and KSPi ⁶ .

In some embodiments, KSPi ⁵ is: ; and KSPi ⁶ is: or ; Wherein: # represents the bond between L ⁴ and KSPi ⁵ ; ## represents the bond between KSPi ⁵ and P ⁵ ; and ### represents the bond between P ⁵ and KSPi ⁶ .

In some embodiments, the antibody drug conjugate is: , , , , , , , , , , , , or .

The antibody-2-drug conjugate of the present invention is obtained by coupling two different payload linker prodrivers to the same antibody in series and applying them twice separately according to the descriptions described in WO2016/207089, WO2017/162663, and WO2018/114798. The obtained A2DC of the present invention comprises two different payload linkers both linked to lysine residues, or two different payload linkers both linked to cysteine residues, or one payload linker linked to a lysine residue and the other payload linker linked to a cysteine residue. Cysteine residues suitable for payload linker attachment can be obtained by reducing interchain disulfide bridges or by engineering cysteine residues into the antibody.

The A2DC of the present invention with different antibodies targeting multiple targets combines different payload characteristics, resulting in an advantageous profile: – Overcoming the great challenge of increasing DAR without encountering aggregate formation, especially due to the polar and hydrophilic KSPi payload. – Impermeable payloads often increase potency due to intracellular accumulation in tumor cells, which is particularly beneficial in vivo when tumor cells display homogeneous target expression. Extremely high potency is achieved when two impermeable KSPi payloads are linked to antibodies, resulting in increased DAR. – Permeable payloads exhibit a bystander effect, which is particularly important when tumors display heterogeneous target expression. – Positive features of the A2DC combination of the present invention provide A2DC that are effective against different cells with a wide variability in target expression profiles and have been shown to be compatible with antibodies that treat different targets.

To further improve the tumor selectivity of A2DC and its metabolites, the antibody conjugates are provided with peptide derivatives that can be released by tumor-associated enzymes such as tadalafil or cathepsin. Thus, the tumor selectivity is determined not only by the selection of the antibody but also by the enzymatic cleavage of the peptide derivative (e.g. by the tumor-associated enzyme tadalafil).

According to the present invention, the peptide derivative may be present in a linker that links the antibody to the KSP inhibitor. Such antibody conjugates are antibody-2-drug conjugates (A2DC) according to the present invention.

The spinodal kinesin inhibitor used according to the invention has an amine group essential for this action. By modifying this amine group with a peptide derivative, the action on spinodal kinesin is blocked and thus also the generation of cytotoxic effects is inhibited. However, if this peptide residue can be released by tumor-related enzymes (such as soy protein), the effect can be reconstructed in a controlled manner in tumor tissue. In this case, the modification of the amine group is not part of the linker. The present invention therefore relates to a conjugate with an inactive prodromal molecule of a KSP inhibitor, which is processed by the tumor-associated lysosomal peptidase tadalafil to give an active metabolite only in a tumor and is thus able to re-display its cytotoxic activity in a controlled manner in a tumor. A conjugate with a KSP inhibitor, wherein the free amine groups are correspondingly blocked, is also referred to as APDC according to the present invention. APDC is particularly preferred.

The antibody-2-drug conjugates of the present invention have enzyme-cleavable peptide sequences ^P3 and ^P4 capable of releasing two active metabolites, which can be flexibly customized according to the desired physicochemical profile. In A2DC of formula (II'), antibody attachment is achieved via a branched linker ^L3 , while in A2DC of formula (III), antibody attachment is achieved via a non-cleavable linker ^L4 connected to ^KSPi5 . (II') (III)

A2DC can be linked to the antibody in a variety of ways, via chemical or enzymatic linking schemes, preferably via a lysine residue or via a cysteine residue. Cysteine residues suitable for efficient cargo linker attachment can be obtained by reducing interchain disulfide bridges or by engineering cysteine residues into the antibody.

The A2DC of the present invention with different antibodies targeting multiple targets combines different payload features, resulting in an advantageous profile: – Overcoming the significant challenge of increasing DAR without encountering aggregate formation, particularly by utilizing polar and hydrophilic KSPi payload and linker combinations. – Impermeable payloads often increase potency due to intracellular accumulation in tumor cells, which is particularly beneficial in vivo when tumor cells display homogeneous target expression. Extremely high potency is achieved when two impermeable KSPi payloads are linked to antibodies, resulting in increased DAR. – Permeable payloads exhibit a bystander effect, which is particularly important when tumors display heterogeneous target expression. – The positive feature of the A2DC combination of the present invention is that it provides A2DC that is effective against different cells with a wide range of target expression profile variability. This shows that this A2DC is compatible with antibodies that treat different targets.

In a specific embodiment of the present invention, the release of both metabolites is mediated by cleavage of the linker by the tumor-associated protease tadalafil. Alternatively, A2DC can also be designed to mediate the release of the effective cargo by other proteases (such as tissue proteases) or by glycosidases (such as β-glucuronidase).

In a preferred embodiment, the target molecule is a selective cancer target molecule. In a particularly preferred embodiment, the target molecule is a protein. In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein. Cancer target molecules are known to those skilled in the art. Examples of such molecules are listed below.

Examples of cancer target molecules are: (1) EGF receptor (NCBI reference sequence NP_005219.2), SEQ ID NO: 213 (1210 amino acids). (2) Mesothelin (SwissProt reference Q13421-3), SEQ ID NO: 214 (622 amino acids). (3) Her2 (NCBI reference sequence NP_004439.2), SEQ ID NO: 218 (4) TWEAKR (SEQ ID NO: 169 protein); SEQ ID NO: 170 (DNA) (5) CXC interleukin receptor CXCR5 (CD185; Gene ID 643)(SwissProt: P32302) (6) Surface receptor IL3RA (CD123; Gene ID: 3561)

In some embodiments, the antibodies of the present invention can be any suitable antibodies. In some embodiments, the antibodies include but are not limited to GUCY2C (guanylate cyclase C), ROR1, HER3, FGFR4, EGFR/PD-L1 bispecific antibodies and B7H4.

Polyclonal antibodies can be prepared by methods known to those skilled in the art. Monoclonal antibodies can be prepared by methods known to those skilled in the art (Köhler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies can be prepared by methods known to those skilled in the art (Olsson et al., Meth Enzymol. 92, 3-16 or U.S. Patent No. 4,816,567 to Cabilly et al. or U.S. Patent No. 4,816,397 to Boss et al.).

Those skilled in the art are aware of various methods for preparing human antibodies and fragments thereof, such as by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(1):65-93) or phage display technology (Clackson et al., Nature. 1991 Aug 15; 352(6336):624-8). The antibodies of the present invention can be obtained from a recombinant antibody library, which is composed of amino acid sequences of a variety of antibodies collected from, for example, many healthy volunteers. Antibodies can also be produced by known recombinant DNA technology. The nucleic acid sequence of the antibody can be obtained by conventional sequencing or from a publicly available database.

In some embodiments, the antibody or antigen-binding fragment thereof (AK) comprises an antibody or antibody region (eg, CDR, variable domain) as shown in the following table: SEQ ID NO. describe sequence 1 TPP-981 VH QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT NYGVHWVRQSPGKGLEWLGV IWSGGNTDYN TPFTSRLSIN KDNSKSQVFF KMNSLQSNDT AIYYCARALT YYDYEFAYWG QGTLVTVSA 2 TPP-981 HCDR1 NYGVH 3 TPP-981 HCDR2 VIWSGGNTDY NTPFTS 4 TPP-981 HCDR3 ALTYYDYEFA Y 5 TPP-981 VL DILLTQSPVI LSVSPGERVS FSCRASQSIG TNIHWYQQRT NGSPRLLIKY ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYCQQ NNNWPTTFGA GTKLELK 6 TPP-981 LCDR1 RASQSIGTNI H 7 TPP-981 LCDR2 YASESIS 8 TPP-981 LCDR3 QQNNNWPTT 9 TPP-981 IgG HC PS SSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQQGNVFSCSVM HEALHNHYTQ KSLSLSPGK 10 TPP-981 IgG LC DILLTQSPVI LSVSPGERVS FSCRASQSIG TNIHWYQQRT NGSPRLLIKY ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYCQQ NNNWPTTFGA GTKLELKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACE VTHQG LSSPVTKSFN RGEC 11 TPP-1015 VH EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 12 TPP-1015 HCDR1 D T 13 TPP-1015 HCDR2 RIYPTNGYTR YADSVKG 14 TPP-1015 HCDR3 WGGDGFYAMD Y 15 TPP-1015 VL DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIK 16 TPP-1015 LCDR1 RASQDVNTAV A 17 TPP-1015 LCDR2 SASFLYS 18 TPP-1015 LCDR3 QQHYTTPPT 19 TPP-1015 IgG HC EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSL GTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 20 TPP-1015 IgG LC DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEK HK VYACEVTHQG LSSPVTKSFN RGEC 31 TPP-2658 VH EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMIWVRQA PGKGLEWVSY ISPSGGSTHY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGGDTYFDYFDYW GQGTLVTVSS A 32 TPP-2658 HCDR1 PYPMI 33 TPP-2658 HCDR2 YISPSGGSTH YADSVKG 34 TPP-2658 HCDR3 GGDTYFDYFD Y 35 TPP-2658 VL DIQMTQSPSS LSASVGDRVT ITCRASQSIS GYLNWYQQKP GKAPKLLIYQ ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTSPFITFG QGTKVEIK 36 TPP-2658 LCDR1 RASQSISGYL N 37 TPP-2658 LCDR2 QASSLQS 38 TPP-2658 LCDR3 QQSYTSPFIT 39 TPP-2658 IgG HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMIWVRQA PGKGLEWVSY ISPSGGSTHY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGGDTYFDYFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQ T YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYA STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 40 TPP-2658 IgG LC DIQMTQSPSS LSASVGDRVT ITCRASQSIS GYLNWYQQKP GKAPKLLIYQ ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTSPFITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 61 TPP-7007 VH QVQLVQSGAE VKKPGASVKV SCKASGYTFT DFIIAWVKQA PGQGLEWIGE IYPGTGRTYY SEKFRGKATL TADTSTSTAY MELSSLRSED TAVYFCARRT IYYDYDGDYW GQGTLVTVSS 62 TPP-7007 HCDR1 DFIIA 63 TPP-7007 HCDR2 EIYPGTGRTY YSEKFRG 64 TPP-7007 HCDR3 RTIYYDYDGD Y 65 TPP-7007 VL IK 66 TPP-7007 LCDR1 RSSKSLLHSN GITYLY 67 TPP-7007 LCDR2 QMSNLAS 68 TPP-7007 LCDR3 AHNLELPWT 69 TPP-7007 IgG HC QVQLVQSGAE VKKPGASVKV SCKASGYTFT DFIIAWVKQA PGQGLEWIGE IYPGTGRTTYY SEKFRGKATL TADTSSTAY MELSSLRSED TAVYFCARRT IYYDYDGDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSL GTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 70 TPP-7007 IgG LC DIVMTQSPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAHNLELP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD Y EKHKVYACE VTHQGLSSPV TKSFNRGEC 71 TPP-2090 VH EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMIWVRQA PGKGLEWVSY ISPSGGSTHY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG DTYFDYFDYW GQGTLVTVSS 72 TPP-2090 HCDR1 PYPMI 73 TPP-2090 HCDR2 YISPSGGSTH YADSVKG 74 TPP-2090 HCDR3 GGDTYFDYFD Y 75 TPP-2090 VL DIQMTQSPSS LSASVGDRVT ITCRASQSIS GYLNWYQQKP GKAPKLLIYQ ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTSPFITFG QGTKVEIK 76 TPP-2090 LCDR1 RASQSISGYL N 77 TPP-2090 LCDR2 QASSLQS 78 TPP-2090 LCDR3 QQSYTSPFIT 79 TPP-2090 IgG HC EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMIWVRQA PGKGLEWVSY ISPSGGSTHY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG DTYFDYFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQ TI LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 80 TPP-2090 IgG LC DIQMTQSPSS LSASVGDRVT ITCRASQSIS GYLNWYQQKP GKAPKLLIYQ ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTSPFITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 81 TPP-170 VH QVELVQSGAE VKKPGESLKI SCKGSGYSFT SYWIGWVRQA PGKGLEWMGI IDPGDSRTRY SPSFQGQVTI SADKSISTAYLQWSSLKASD TAMYYCARGQLYGGTYMDGWGQGTLVTVSS 82 TPP-170 HCDR1 SYWIG 83 TPP-170 HCDR2 IIDPGDSRTR YSPSFQG 84 TPP-170 HCDR3 GQLYGGTYMD G 85 TPP-170 VL DIALTQPASV SGSPGQSITI SCTGTSSDIG GYNSVSWYQQHPGKAPKLMI YGVNNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYDIESATP VFGGGTKLTV L 86 TPP-170 LCDR1 TGTSSDIGGY NSVS 87 TPP-170 LCDR2 GVNNRPS 88 TPP-170 LCDR3 SSYDIESATP V 89 TPP-170 IgG HC QVELVQSGAE VKKPGESLKI SCKGSGYSFT SYWIGWVRQA PGKGLEWMGI IDPGDSRTRY SPSFQGQVTI SADKSISTAYLQWSSLKASD TAMYYCARGQLYGGTYMDGWGQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT Y ICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 90 TPP-170 IgG LC DIALTQPASV SGSPGQSITI SCTGTSSDIG GYNSVSWYQQHPGKAPKLMI YGVNNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYDIESTP VFGGGTKLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKGDSSPV KAGVETTTPS KQSNNKYAAS SYLSLTPEQW KS HRSYSCQV THEGSTVEKT VAPTECS 91 TPP-9476 VH QVQLVQSGAEVKKPGSSVKV SCKASGGTFS DYYMKWVRQAPGQGLEWMGD IIPSNGATFY NQKFKGRVTITADESTSTAY MELSSLRSEDTAVYYCARSH LLRASWFAYW GQGTLVTVSS 92 TPP-9476 HCDR1 DYYMK 93 TPP-9476 HCDR2 DIIPSNGATF YNQKFKG 94 TPP-9476 HCDR3 SHLLRASWFA Y 95 TPP-9476 VL DIVMTQSPDS LAVSLGERAT INCESSQSVL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 96 TPP-9476 LCDR1 ESSQSVLNSG NQKNYLT 97 TPP-9476 LCDR2 WASTRES 98 TPP-9476 LCDR3 QNDYSYPYT 99 TPP-9476 IgG HC QVQLVQSGAEVKKPGSSVKV SCKASGGTFS DYYMKWVRQAPGQGLEWMGD IIPSNGATFY NQKFKGRVTITADESTSTAYMELSSLRSED TAVYYCARSH LLRASWFAYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 100 TPP-9476 IgG LC DIVMTQSPDS LAVSLGERAT INCESSQSVL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKA DY EKHKVYAC EVTHQGLSSP VTKSFNRGEC 101 TPP-9574 VH EVQLVESGGG LIQPGGSLRL SCAASGFTFS TSGMHWFRQAPGKGLEWVAY ISSSSGFVYA DAVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARSEA AFWGQGTLVT VSS 102 TPP-9574 HCDR1 TSGMH 103 TPP-9574 HCDR2 YISSSSGFVY ADAVKG 104 TPP-9574 HCDR3 SEAAF 105 TPP-9574 VL DIVMTQSPLS LPVTPGEPAS ISCRSQKSRL SRMGITPLNW YLQKPGQSPQ LLIYRMSNLA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQFLEYP PTFGQGTKLE IK 106 TPP-9574 LCDR1 RSQKSRLSRM GITPLN 107 TPP-9574 LCDR2 RMSNLAS 108 TPP-9574 LCDR3 AQFLEYPPT 109 TPP-9574 IgG HC EVQLVESGGG LIQPGGSLRL SCAASGFTFS TSGMHWFRQA PGKGLEWVAY ISSSSGFVYA DAVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARSEA AFWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNV NH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PG 110 TPP-9574 IgG LC DIVMTQSPLS LPVTPGEPAS ISCRSQKSRL SRMGITPLNW YLQKPGQSPQ LLIYRMSNLA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQFLEYP PTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSK AD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 111 TPP-6013 VH QVQLQESGPG LVKPSQSLSL TCSVTDYSIT SGYYWNWIRQFPGNKLEWMG YISYDGSNNY NPSLKNRISI TRDTSKNQFF LKLSSVTTED TATYYCSRGE GFYFDSWGQG TTLTVSS 112 TPP-6013 HCDR1 SGYYW 113 TPP-6013 HCDR2 YISYDGSNNY NPSLKN 114 TPP-6013 HCDR3 GEGFYFDS 115 TPP-6013 VL DIMMSQSPSS LAVSVGEKFT MTCKSSQSLF FGSTQKNYLA WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFTLA ISSVMPEDLA VYYCQQYYNY PWTFGGGTKL EIK 116 TPP-6013 LCDR1 KSSQSLFFGS TQKNYLA 117 TPP-6013 LCDR2 WASTRES 118 TPP-6013 LCDR3 QQYYNYPWT 119 TPP-6013 IgG HC QVQLQESGPG LVKPSQSLSL TCSVTDYSIT SGYYWNWIRQFPGNKLEWMG YISYDGSNNY NPSLKNRISI TRDTSKNQFF LKLSSVTTED TATYYCSRGE GFYFDSWGQG TTLTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSL GTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 120 TPP-6013 IgG LC DIMMSQSPSS LAVSVGEKFT MTCKSSQSLF FGSTQKNYLA WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFTLA ISSVMPEDLA VYYCQQYYNY PWTFGGGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNALQSGNSQESVT EQDSKDSTYS LSSTLT LSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 2; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 3; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 4; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 6; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 7; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 8.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 12; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 13; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 14; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 32; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 33; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 34; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 62; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 63; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 64; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 68.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 72; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 73; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 74; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 78.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 82; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 83; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 84; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 92; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 93; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 94; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 96; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 97; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 98.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 106; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 107; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 108.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 112; b) a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 113; c) a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 114; d) a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 116; e) a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 117; and/or f) a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 118.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 9; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 10.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 11; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 19; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 31; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 39; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 40.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 61; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 65. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 69; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 70. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 69; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 70.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 71; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 75. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 71; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 75.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 79; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 80. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 79; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 80.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 81; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 85. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 81; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 89; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 90. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 89; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 90.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 91; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 95. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 99; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 100. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 100.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 101; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 105. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 109; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 110. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 109; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 110.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 111; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 115. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 119; and/or b) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 120. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 119; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 120.

The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the reference polypeptide sequence with the candidate sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. Definition

Unless otherwise defined, all technical terms, notations, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the art.

Throughout this application, various embodiments may be presented in range format. It should be understood that descriptions in range format are for convenience and brevity only and should not be construed as fixed limitations on the scope of the invention. Therefore, descriptions of ranges should be considered to have specifically disclosed all possible subranges and individual numbers within such ranges. For example, descriptions of ranges such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within such ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

As used in this specification and claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. For example, the term "a sample" includes plural samples, including mixtures thereof.

The terms "determine," "measure," "evaluate," "assess," "assay," and "analyze" are generally used interchangeably herein to refer to forms of measurement. Such terms include determining whether an element is present (e.g., detecting). Such terms may include quantitative, qualitative, or both quantitative and qualitative measurements. Assessments may be relative or absolute. "Detecting the presence of" may include determining the amount of something present, in addition to determining whether it is present, depending on the context.

The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" may be a biological entity containing the expressed genetic material. The biological entity may be a plant, an animal, or a microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject may be tissues, cells, and progeny thereof, of a biological entity obtained in vivo or cultured in vitro. The subject may be a mammal. The mammal may be a human. The subject may be diagnosed or suspected to be at high risk for a disease. In some cases, the subject may not be diagnosed or suspected to be at high risk for a disease.

As used herein, the term "about" a number refers to the number plus or minus 15% of the number. The term "about" a range refers to the range minus 15% of the lower limit and plus 15% of the upper limit.

As used herein, the term "treatment" or "treating" is used in reference to a medical regimen or other intervention regimen for obtaining a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and/or preventive benefit. Therapeutic benefit may refer to the eradication or improvement of the symptoms or underlying condition being treated. In addition, therapeutic benefit may also be achieved by eradicating or improving one or more of the physiological symptoms associated with the underlying condition, such that improvement is observed in the subject, although the subject may still suffer from the underlying condition. Preventive effects include delaying, preventing or eliminating the appearance of a disease or condition; delaying or eliminating the onset of symptoms of a disease or condition; slowing, stopping or reversing the progression of a disease or condition; or any combination thereof. For preventive benefit, subjects at risk for a particular disease or reporting one or more of the physiological symptoms of a disease may be treated even though the disease may not have been diagnosed.

Cancer is the result of uncontrolled cell growth in most diverse tissues. In many cases, new cells infiltrate existing tissues (invasive growth), or they metastasize to distant organs. Cancer arises in many different organs and often has a tissue-specific course. Therefore, the term "cancer" is a general term that describes a large group of defined diseases of different organs, tissues, and cell types.

The term "cancer target molecule" describes a target molecule that is present in greater abundance on one or more cancer cell species than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species compared to non-cancer cells of the same tissue type, wherein selectively describes an enrichment on cancer cells that is at least two-fold greater than on non-cancer cells of the same tissue type ("selective cancer target molecule"). The use of a cancer target molecule allows for the selective treatment of cancer cells using the conjugates according to the invention.

In the broadest sense, "target molecule" is understood to mean a molecule present in a target cell population and can be a protein (e.g. a receptor for a growth factor) or a non-peptide molecule (e.g. a sugar or a phospholipid). It is preferably a receptor or an antigen.

The term "extracellular" target molecule describes a target molecule that is located outside the cell, or a part of a target molecule that is located outside the cell, i.e. a binder that can bind to its extracellular target molecule on an intact cell. The extracellular target molecule can be anchored in the cell membrane or be a component of the cell membrane. The person skilled in the art knows methods for identifying extracellular target molecules. For proteins, this can be done by determining the transmembrane domain and the orientation of the protein in the membrane. Such data are usually deposited in protein databases (e.g. SwissProt).

According to the present invention, the term "antibody" is understood in its broadest sense and includes immunoglobulin molecules, such as complete or modified monoclonal antibodies, polyclonal antibodies or multispecific antibodies (such as bispecific antibodies). Immunoglobulin molecules preferably include molecules with four polypeptide chains: two heavy chains (H chains) and two light chains (L chains), which are usually connected by disulfide bridges. Each heavy chain includes a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. The heavy chain constant domain can, for example, include three domains CH1, CH2 and CH3. Each light chain includes a variable domain (abbreviated as VL) and a constant domain. The light chain constant domain includes a domain (abbreviated as CL). The VH and VL domains can be further subdivided into regions with high variability, also known as complementation determining regions (abbreviated as CDR), and regions with low sequence variability (framework regions, abbreviated as FR). Typically, each VH and VL region consists of three CDRs and up to four FRs. For example, from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody can be obtained from any suitable species, such as rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. The antibody can be, for example, a human antibody, a humanized antibody or a chimeric antibody.

The term "monoclonal" antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies in the population are identical except for possible rare mutations that occur naturally. Monoclonal antibodies recognize a single antigen binding site with high specificity. The term monoclonal antibody does not refer to a specific method of preparation.

Depending on the amino acid sequence of the heavy chain constant domain, antibodies can be classified into different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of them can be divided into other subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes are called [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ], and [my/μ]. Both the three-dimensional structure and the subunit structure of antibodies are known.

The term "intact" antibody refers to an antibody comprising an antigen binding domain and light and heavy chain cognate domains. The cognate domains may be naturally occurring domains or variants thereof with various modified amino acid positions, and may also be glycosylated.

The term "modified intact" antibody refers to an intact antibody in which the amino or carboxyl terminus is fused to another polypeptide or protein not derived from the antibody by means of a covalent bond (e.g., a peptide bond). In addition, the antibody can be modified so that reactive cysteine is introduced at a defined position to facilitate coupling with a toxin group (see Junutula et al., Nat Biotechnol. 2008 Aug; 26(8):925-32).

"Amino acid modification" or "mutation" herein means an amino acid substitution, insertion and/or deletion in a polypeptide sequence. The preferred amino acid modification herein is substitution. "Amino acid substitution" or "substitution" herein means replacing an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W describes a variant of a parent polypeptide in which the tyrosine at position 50 is replaced with tryptophan. A "variant" of a polypeptide describes a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide (usually a native or "parent" polypeptide). Polypeptide variants may have one or more amino acid substitutions, deletions and/or insertions at specific positions in the native amino acid sequence.

The term "human" antibody refers to an antibody that can be obtained from humans or an antibody that is a synthetic human antibody. A "synthetic" human antibody is an antibody that can be obtained partially or completely from a synthetic sequence by computer simulation based on analysis of human antibody sequences. Human antibodies can be encoded by nucleic acids isolated, for example, from a library of antibody sequences derived from humans. Examples of such antibodies can be found in Soderlind et al., Nature Biotech. 2000, 18: 853-856. Such "human" and "synthetic" antibodies also include aglycosylated variants that have been produced by deglycosylation with PNGaseF or by mutating N297 (Kabat numbering) of the rechain to any other amino acid.

The term "humanized" or "chimeric" antibody describes an antibody composed of non-human and human portions of sequence. In these antibodies, a portion of the sequence of a human immunoglobulin (acceptor) is replaced by a portion of the sequence of a non-human immunoglobulin (donor). In many cases, the donor is a murine immunoglobulin. In the case of humanized antibodies, the acceptor CDR amino acids are replaced by donor amino acids. Sometimes, framework amino acids are also replaced by corresponding amino acids from the donor. In some cases, humanized antibodies contain amino acids that are not present in either the acceptor or the donor, which are introduced during antibody optimization. In the case of chimeric antibodies, the variable domains of the donor immunoglobulin are fused to the constant regions of a human antibody. Such "humanized" and "chimeric" antibodies also include aglycosylated variants that have been produced by deglycosylation with PNGaseF or by mutating recombinant chain N297 (Kabat numbering) to any other amino acid.

As used herein, the term "complementary determining region (CDR)" refers to those amino acids of an antibody variable domain that are required for binding to an antigen. Typically, each variable region has three CDR regions, referred to as CDR1, CDR2, and CDR3. Each CDR region may contain amino acids according to the Kabat definition and/or hypervariable cyclic amino acids according to the Chotia definition. The definition according to Kabat includes, for example, the region of approximately amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) of the variable light chain/domain (VL) and the region of approximately amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) of the variable heavy chain/domain (VH) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). According to the definition of Chotia, the region of about amino acid positions 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) of the variable light chain (VL) and the region of about amino acid positions 26-32 (CDR1), 53-55 (CDR2) and 96-101 (CDR3) of the variable heavy chain (VH) are included (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases, the CDR may include amino acids from the CDR region defined according to Kabat and Chotia.

The term "functional fragment" or "antigen-binding antibody fragment" of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g., the variable domain of IgG) that still contains the antigen-binding domain of the antibody/immunoglobulin. The "antigen-binding domain" of an antibody generally comprises one or more hypervariable regions of the antibody, such as the CDR, CDR2 and/or CDR3 regions. However, the "framework" or "skeleton" regions of an antibody may also play a role during the binding of the antibody to the antigen. The framework regions form the framework of the CDRs. Preferably, the antigen-binding domain comprises at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably comprises amino acids 3 to 107 of the variable light chain and amino acids 4 to 111 of the variable heavy chain, and even more preferably comprises the complete variable light chain and heavy chain, i.e., amino acids 1 to 109 of VL and amino acids 1 to 113 of VH (according to WO97/08320 numbering).

The "functional fragment" or "antigen-binding antibody fragment" of the present invention non-definitive includes Fab, Fab', F(ab')2 and Fv fragments, bifunctional antibodies (diabodies), single domain antibodies (DAbs), linear antibodies, individual antibody chains (single chain Fv, abbreviated as scFv); and multispecific antibodies, such as bispecific and trispecific antibodies formed from, for example, antibody fragments, C. A. K Borrebaeck (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann and S. Duebel (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag. Antibodies other than "multispecific" or "multifunctional" antibodies are those antibodies having the same binding site. Multispecific antibodies may be specific for different antigenic determinants of an antigen or may be specific for antigenic determinants of more than one antigen (see, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., 1991, J. Immunol. 14760 69; U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819; or Kostelny et al., 1992, J. Immunol. 148 1547 1553). The F(ab')2 or Fab molecule can be constructed such that the number of intermolecular disulfide interactions between the Ch1 and CL domains is reduced or completely prevented.

"Antigenic determinant" refers to a protein determinant that is able to specifically bind to an immunoglobulin or T-cell receptor. Antigenic determinants are usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains or combinations thereof) and usually have specific 3D structural characteristics and specific charge characteristics.

"Functional fragments" or "antigen-binding antibody fragments" can be fused to another polypeptide or protein not derived from the antibody via its amino or carboxyl terminus by means of a covalent bond (e.g., a peptide bond). In addition, antibodies and antigen-binding fragments can be modified by introducing reactive cysteine at defined positions to facilitate coupling with toxin groups (see Junutula et al., Nat Biotechnol. 2008 Aug; 26(8):925-32).

An "isolated" antibody or conjugate has been purified to remove other components of the cells. Contaminating components of the cells that may interfere with diagnostic or therapeutic uses are, for example, enzymes, hormones, or other peptide or non-peptide components of the cells. Preferred antibodies or conjugates are purified to a degree of purity greater than 95% by weight of the antibody or conjugate (e.g., as determined by the Lowry method, UV-Vis spectroscopy, or by SDS capillary gel electrophoresis). Alternatively, the antibody may be purified to the extent that at least 15 amino acids of the amino-terminal or internal amino acid sequence can be determined or to the extent that homogeneity is determined by SDS-PAGE under reducing or non-reducing conditions (which may be detected by Coomassie Blue staining or, preferably, by silver staining). However, antibodies are usually prepared by one or more purification steps.

The term "specific binding" or "binding specifically" refers to an antibody or binder that binds to a predetermined antigen/target molecule. Specific binding of an antibody or binder typically describes an antibody or binder with an affinity (in the form of a Kd value) of at least 10 ^{− 7} M; i.e., it preferably has a Kd value of less than 10 ^{− 7} M, and an affinity of the antibody or binder for the predetermined antigen/target molecule that is at least twice as high as that for a non-specific antigen/target molecule (e.g., bovine serum albumin or casein) that is not the predetermined antigen/target molecule or a closely related antigen/target molecule. Specific binding of an antibody or binder does not exclude binding of the antibody or binder to multiple antigens/target molecules (e.g., heterologs of different species). The antibody preferably has an affinity (in the form of a Kd value; in other words, it preferably has a Kd value of less than ^10-7 M), preferably at least ^10-9 M, more preferably in the range of ^10-9 M to ^10-11 M. The Kd value can be determined, for example ^, by surface plasmon resonance spectroscopy.

Alkyl is a straight or branched chain saturated monovalent hydrocarbon group having 1 to 10 carbon atoms (C ₁ -C ₁₀ alkyl), generally 1 to 6 carbon atoms (C ₁ -C ₆ alkyl), preferably 1 to 4 carbon atoms (C ₁ -C ₄ alkyl) and more preferably 1 to 3 carbon atoms (C ₁ -C ₃ alkyl).

Preferred examples include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, dibutyl, tertiary butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl and 1,2-dimethylbutyl.

Heteroalkyl is a straight and/or branched alkyl chain having 1 to 10 carbon atoms and may be doped once or more than once with one or more of the following groups: -O-, -S-, -C(═O)-, -S(═O)-, -S(═O) ₂ -, -NR ^y -, -NR ^y C(═O)-, -C(═O)-NR ^y -, -NR ^y NR ^y -, -S(═O) 2 -NR ^y NR ^y -, -C(═O)-NR ^y NR ^y -, -CR ^x ═NO-, and the alkyl chain including the side chain (if present) may be doped with -NH-C(═O)-NH ₂ , -C(═O)-OH, -OH, -NH ₂ , -NH-C(═NNH ₂ )-, sulfonamide, sulfonate, sulfone or sulfonic acid substituted.

In this context, R ^y is in each case -H, phenyl, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl or C ₂ -C ₁₀ alkynyl, which in turn may in each case be substituted by -NH-C(═O)-NH ₂ , -C(═O)-OH, -OH, -NH ₂ , -NH-C(═NNH ₂ )-, sulfonamide, sulfone, sulfone or sulfonic acid.

In this context, R ^x is -H, C ₁ -C ₃ alkyl or phenyl.

Alkenyl is a straight or branched monovalent hydrocarbon chain having one or two double bonds and 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms ( _C2 - _C10 alkenyl), in particular 2 or 3 carbon atoms ( _C2 - _C3 alkenyl), wherein, as will be apparent, when the alkenyl contains more than one double bond, the double bonds may be separated from one another or bonded to one another. Alkenyl is, for example, ethenyl, prop-2-en-1-yl (or "allyl"), prop-1-en-1-yl, but-3-enyl, but-2-enyl, but-1-enyl, pent-4-enyl, pent-3-enyl, pent-2-enyl, pent-1-enyl, hex-5-enyl, hex-4-enyl, hex-3-enyl, hex-2-enyl, hex-1-enyl, prop-1-en-2-yl (or "isopropenyl"), 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, 1-methyl prop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, 2-methylbut-2-enyl, 1-methylbut-2-enyl, 3-methylbut-1-enyl, 2-methylbut-1-enyl, 1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl, 4-methylpent-4-enyl, 3-methylpent-4-enyl, 2-methylpent-4-enyl, 1-methylpent-4-enyl, 4-methylpent-3-enyl, 3-methylpent-3-enyl, 2-methylpent-3-enyl, 1-methylpent-3-enyl, 4-methylpent-2-enyl, 3-methylpent-2-enyl, 2-methylpent-2-enyl, 1-methylpent-2-enyl, 4-methylpent-1-enyl, 3-methylpent-1-enyl, 2-methylpent-1-enyl, 1-methylpent-1-enyl, 3-ethylbut-3-enyl, 2-ethylbut-3-enyl, 1-ethylbut-3-enyl, 3-ethylbut-2-enyl, 2-ethylbut-2-enyl, 1-ethylbut The group may be any of a vinyl group, a 1-propyl group, a 2-isopropyl group, a 1-isopropyl group, a 2-propyl group, a 1-propyl group, a 2-isopropyl group, a 1-isopropyl group, a 3,3-dimethyl group, a 1-(1,1-dimethylethyl)vinyl group, a buta-1,3-dienyl group, a penta-1,4-dienyl group or a hexa-1,5-dienyl group. More specifically, the group may be a vinyl group or an allyl group.

Alkynyl is a straight or branched monovalent hydrocarbon chain having one radical and 2, 3, 4, 5 _, 6, 7, 8, 9 or 10 carbon atoms (C ₂ -C ₁₀ alkynyl), in particular 2 or 3 carbon atoms (C ₂ -C ₃ alkynyl). The _6- alkynyl group is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1-methylprop-2-ynyl, 2-methylbut-3-ynyl, 1-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut-1-ynyl, 1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methyl In some embodiments, the alkynyl radical is pent-4-ynyl, 1-methylpent-4-ynyl, 2-methylpent-3-ynyl, 1-methylpent-3-ynyl, 4-methylpent-2-ynyl, 1-methylpent-2-ynyl, 4-methylpent-1-ynyl, 3-methylpent-1-ynyl, 2-ethylbut-3-ynyl, 1-ethylbut-3-ynyl, 1-ethylbut-2-ynyl, 1-propylprop-2-ynyl, 1-isopropylprop-2-ynyl, 2,2-dimethylbut-3-ynyl, 1,1-dimethylbut-3-ynyl, 1,1-dimethylbut-2-ynyl or 3,3-dimethylbut-1-ynyl. More specifically, the alkynyl radical is ethynyl, prop-1-ynyl or prop-2-ynyl.

Some of the headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Examples

The following illustrative examples represent embodiments of the stimulation, systems and methods described herein and are not intended to be limiting in any way. Part I: Preparation and Characterization of Compounds of Formula I and Synthetic Antibody -2- Drug Conjugates (A2DCs) B-1. General Methods for Expressing Antibodies

As described in Tom et al., Chapter 12 of Methods Express: Expression Systems, ed. Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007, a DNA sequence encoding the protein sequence (amino acid sequence) of the antibody used (e.g., TPP-981, TPP-1015, TPP-7007, TPP-2658, TPP-2090, TPP-9476, and TPP-9574) is inserted into a transient expression vector. B-2 General methods for expressing antibodies in mammalian cells

Antibodies such as TPP-981, TPP-1015, TPP-7007, TPP-2658, TPP-2090, TPP-9476 and TPP-9574 are produced in transient mammalian cell culture as described in Tom et al., Chapter 12 of Methods Express: Expression Systems, ed. Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. B-3 General method for purification of antibodies from cell supernatants

Antibodies, such as TPP-981, TPP-1015, TPP-7007, TPP-2658, TPP-2090, TPP-9476 and TPP-9574, were obtained from cell culture supernatants. The cell supernatants were clarified by centrifugation of the cells. The cell supernatants were then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. For this purpose, the column was equilibrated in DPBS (pH 7.4) (Sigma/Aldrich), the cell supernatant was applied, and the column was washed with approximately 10 column volumes of DPBS (pH 7.4) + 500 mM sodium chloride. Antibodies were eluted in 50 mM sodium acetate (pH 3.5) + 500 mM sodium chloride and further purified by gel filtration on a Superdex 200 column (GE Healthcare) in DPBS (pH 7.4).

Commercial antibodies were purified from commercial products by standard chromatographic methods (protein A chromatography, preparative gel filtration chromatography (SEC-size exclusion chromatography)). General procedure for cysteine coupling

The following antibodies were used in the exemplary coupling schemes described in the A2DC Examples section: Example a: Anti-EGFR mAb (cetuximab) (TPP-981) Example e: Anti-Her2 mAb (TPP-1015) Example k: Anti-TWEAKR mAb (TPP-7007) Example k: Anti-TWEAKR mAb (TPP-2658) Example k: Anti-TWEAKR mAb (TPP-2090) Example c: Anti-CD123 mAb (TPP-9476) Example x: Anti-CXCR5 mAb (TPP-9574)

To obtain the thiol nucleophile, the antibody can be reduced by the state of the art method also employed in the examples described below. Alternatively, free cysteine residues can be site-specifically engineered into the antibody. Coupling reactions with the electrophiles described in the section "ADC Prodriver Molecules" are typically performed under argon as described in the general procedure in the section "A2DC Examples".

In the bioassay, the concentration of the final A2DC sample was adjusted to a range of 0.5 to 15 mg/ml by re-dilution as necessary. The respective protein concentrations in the A2DC solutions described in the examples were determined. In addition, the antibody loading (drug/mAb ratio) was determined using the methods described herein.

Depending on the linker, the A2DC shown in the examples may also be present in lower or higher levels in the hydrolyzed succinylamide form linked to the antibody.

Specifically, by connecting sub-substructures KSP-I-A2DC linked to the thiol group of the antibody can optionally also be hydrolyzed after coupling by rebuffering and stirring at pH 8 for about 20 to 24 h to obtain A2DC linked via the open chain succinamide.

#1 indicates the sulfide bridge to the antibody, and #2 indicates the point of attachment to the modified KSP inhibitor.

Such linkers, A2DC linked to the antibody via hydrolyzed unsuccinamide, show higher stability against deconjugation by retro-Michael reaction and can be prepared as shown in the following scheme and described in General Procedure A (Lerchen et al., Angew. Chem. Int. Ed. 2018, 57, 15243):

Other potentially hydrolysis-sensitive thianylsuccinamide bridges to the antibody may contain the following linker substructures, where #1 represents the thioether bond to the antibody and #2 represents the point of attachment to the modified KSP inhibitor:

In the structural formula described in the examples, AK ₁ may mean: Example a: anti-EGFR mAb (cetuximab) (TPP-981), partially reduced Example e: anti-Her2 mAb (TPP-1015), partially reduced Example k-7007: anti-TWEAKR mAb (TPP-7007), partially reduced Example k-2658: anti-TWEAKR mAb (TPP-2658), partially reduced Example k-2090: anti-TWEAKR mAb (TPP-2090), partially reduced Example c: anti-CD123 mAb (TPP-9476), partially reduced Example x: anti-CXCR5 mAb (TPP-9574), partially reduced wherein § ¹ represents the bond to the succinimidyl group or to any isomeric hydrolytically releasable succinimidyl group or the alkylene group derived therefrom, and S S represents the sulfur atom of the cysteine residue of the partially reduced antibody. General Procedure for Lysine Coupling

The following antibodies were used in the exemplary coupling schemes described in the A2DC Examples section: Example a: Anti-EGFR mAb (cetuximab) (TPP981) Example e: Anti-Her2 mAb (TPP1015) Example k: Anti-TWEAKR mAb (TPP-7007) Example k: Anti-TWEAKR mAb (TPP-2658) Example k: Anti-TWEAKR mAb (TPP-2090) Example c: Anti-CD123 mAb (TPP-9476) Example x: Anti-CXCR5 mAb (TPP-9574)

In the structural formulas described in the examples, AK ₂ may mean: Example a: anti-EGFR mAb (cetuximab) (TPP981) Example e: anti-Her2 mAb (TPP1015) Example k-7007: anti-TWEAKR mAb (TPP-7007) Example k-2658: anti-TWEAKR mAb (TPP-2658) Example k-2090: anti-TWEAKR mAb (TPP-2090) Example c: anti-CD123 mAb (TPP-9476) Example x: anti-CXCR5 mAb (TPP-9574) wherein § ² represents the bond to the carbonyl group, and NH represents the pendant amine group in the lysine residue of the antibody. Analytical Characterization of A2DC by SEC-UV for Drug Loading and Purity Assessment

Size exclusion chromatography (SEC) was used to determine the drug-antibody ratio (DAR) and purity of each A2DC. 50 μL of A2DC solution was used for analysis. SEC analysis was performed on a 1200 HPLC system monitored at 260 nm and 280 nm. A Superdex 200 10/300 GL column (GE Healthcare) was operated at a flow rate of 0.5 mL/min using phosphate-buffered saline (PBS) with an isocratic gradient at room temperature. The dimer and aggregate content was determined from the integration of the UV peaks of monomers, dimers, and aggregates. To calculate the DAR, the area under the SEC peak curve at 260 nm (A _drug ) and 280 nm (A ₂₈₀ ) (the sum of aggregates, dimers, and monomers) was calculated using the following equation: (Ab = antibody, D = drug, ldrug = wavelength of interest for the drug (260 nm for the KSP toxin group), ε = absorption coefficient, A = absorption at a specific wavelength)

For toxin boluses, experimentally determined absorption coefficients are used. For all antibodies, a set of common absorption coefficients is used (see table below). Absorption coefficients of antibodies and drugs . Wavelength(nm) antibody KSP 280 ε _Ab ²⁸⁰ 0.2284 ε _drug ²⁸⁰ 0.010 260 ε _Ab ²⁶⁰ 0.1163 ε _drug ²⁶⁰ 0.014 Determination of A2DC concentration

The concentration of each A2DC ( _CA2DC ) was determined by measuring the absorption at 280 nm. The concentration was calculated using the absorption coefficient of the respective antibody. The concentration was corrected to take into account the absorbance of the toxin bolus at 280 nm using the following equation: _CA2DC = initial concentration / (1 + DAR _UV * [εdrug _{280 nm} / _{εAntibody 280 nm} ]) where initial concentration = concentration calculated using only the extinction coefficient of the antibody, DAR _UV = drug loading of the respective A2DC determined by UV absorption, εdrug _{280 nm} = extinction coefficient of drug at 280 nm, and εAntibody _{280 nm} = extinction coefficient of antibody at 280 nm). Characterization of A2DC by mass spectrometry

As an alternative to characterization of antibodies and toxigenic species and for DAR determination, ADC and A2DC have been characterized by mass spectrometry.

Molecular weight analysis was performed using a combination of HPLC and ESI-Q-TOF consisting of an I-class HPLC (Waters) for sample desalting and separation, and an Impact HD mass spectrometer (Bruker Daltonik, Bremen) equipped with instrument control and acquisition software HyStar 3.2, ESI Compass 1.7, and Maximum Entropy Deconvolution Option for MS analysis.

Chromatographic system and conditions : Column: Acquity UPLC BEH300C4 1.7 µm, 1.0×50 mm; Column temperature: 70°C; Flow rate: 200 µl/min; Mobile phase solution A: 0.1% formic acid, 94.9% water, 5% ACN; Mobile phase solution B: 0.1% formic acid, 9.9% water, 10% ACN and 80% 2-propanol.

Binary gradient profile of intact antibody : 2min 5% B, 2.5min 50% B, 3.5min 50% B, 5min 95% B, 5.1min 5% B, 5.6min 95% B, 5.7min 5% B, 6.2min 95% B, 6.3min 5% B, 7.5min 5% B

Binary gradient profile of reduced antibody : 2 min 5% B, 4 min 30% B, 5 min 50% B, 7.5 min 50% B, 8.5 min 95% B, 8.6 min 5% B, 9.1 min 95% B, 9.3 min 5% B, 9.8 min 95% B, 9.9 min 5% B, 12 min 5% B

For Cys- coupled ADC and A2DC , the determination of the molecular weight of the individual conjugate species has been measured after deglycosylation and reduction. Dilute approximately 160 pmol of conjugate with 20 mM sodium phosphate (pH 6.5) in a final volume of 25 µl. Add 1 µl PNGase F and incubate the sample overnight at 37°C with gentle shaking (Thermomixer). Denature 10 µl of the deglycosylated sample with 20 µl 5M Gu*HCl (guanidine hydrochloride in 50 mM triethylammonium bicarbonate) at 60°C by boiling the sample for 5 min. To reduce the protein, add 0.5 µl of 255 mM DTT dissolved in water and incubate the mixture at 60°C for approximately 10 min. The cooled samples were acidified with 2 µl of 10% formic acid/water and then analyzed by mass spectrometry as described above. For DAR determination, all spectra relative to the signal in the TIC (total ion chromatogram) were summed and the molecular weights of the different conjugate species were calculated based on MaxEnt decyclization of the light and heavy chains. The average loading of antibodies with phoretic groups was calculated based on the peak area determined by integrating twice the sum of the HC loading and the LC loading, where the HC loading was the sum of the phoretic group number weighted integration results of all heavy chain (HC) peaks divided by the sum of the individual weighted integration results of the HC peaks, and the LC loading was the sum of the phoretic group number weighted integration results of the light chain (LC) peaks divided by the sum of the individual weighted integration results of all LC peaks. For A2DC, the DAR was calculated for each phoretic group species separately. The total DAR was calculated as the sum of the individual DARs of the two phoretic group species.

For Lys- coupled ADC and A2DC , the determination of the molecular weight of the individual conjugate species has been measured after deglycosylation. Dilute approximately 160 pmol of conjugate with 20 mM sodium phosphate, pH 6.5, to a final volume of 25 µl. Add 1 µl PNGase F and incubate the sample overnight at 37°C with gentle shaking (Thermomixer). Acidify 5 µl of the deglycosylated sample with 2 µl of 10% formic acid/water and then dilute to a concentration of 1 pmol/µl with 0.1% formic acid/water. Analyze 3 µl by mass spectrometry as described above. For DAR determination, all spectra relative to the signal in the TIC (Total Ion Chromatography) were summed and the molecular weights of the different conjugate species were calculated based on MaxEnt decyclization of intact antibodies with different numbers of phoretic groups. The DAR was calculated by dividing the sum of the peak areas weighted by the number of phoretic groups by the sum of the unweighted peak areas. For A2DC, the DAR was calculated for each phoretic group species separately. The total DAR was calculated as the sum of the individual DARs of the two phoretic groups species.

For A2DC consisting of a combination of Cys- and Lys -coupled toxin groups , the molecular weight of the individual conjugate species was measured after deglycosylation and reduction. Approximately 160 pmol of conjugate was diluted with 20 mM sodium phosphate (pH 6.5) in a final volume of 25 µl. 1 µl PNGase F was added and the sample was incubated overnight at 37°C with gentle shaking (Thermomixer). 10 µl of the deglycosylated sample was denatured by 20 µl 5M Gu*HCl (guanidine hydrochloride in 50 mM triethylammonium bicarbonate) and boiling at 60°C for approximately 5 min. To reduce the protein, 0.5 µl 255 mM DTT dissolved in water was added and the mixture was incubated at 60°C for approximately 10 min. The cooled samples were acidified with 2 µl of 10% formic acid/water and then analyzed by mass spectrometry as described above. DAR determinations were performed as described for Cys-coupled ADCs. The average loading of the light and heavy chains was calculated for each individual toxin group and/or combination of two toxin groups. The total DAR was calculated by summing the average loading of both the light and heavy chains twice. Examination of antigen binding of A2DC

After coupling has occurred, the ability to bind to the target molecule is checked. Those skilled in the art are familiar with various methods that can be used for this purpose; for example, the affinity of the conjugate can be checked using ELISA technology or surface plasmon resonance analysis (BIA-core™ measurement). Those skilled in the art can measure the concentration of the conjugate using conventional methods, such as measuring antibody conjugates by protein assays (see also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Poison et al., Blood 2007; 1102:616-623). Preferred antibodies and antigen-binding fragments used in A2DCS Table 1 : Protein sequences of antibodies: Antibody TPP-XXX antigen SEQ ID NO: VH SEQ ID NO: H-CDR1 SEQ ID NO: H-CDR2 SEQ ID NO: H-CDR3 SEQ ID NO: VL SEQ ID NO: L-CDR1 SEQ ID NO: L-CDR2 SEQ ID NO: L-CDR3 SEQ ID NO: IgG heavy chain SEQ ID NO: IgG light chain TPP-981 EGFR 1 2 3 4 5 6 7 8 9 10 TPP-1015 HER2 11 12 13 14 15 16 17 18 19 20 TPP-2658 TWEAKR 31 32 33 34 35 36 37 38 39 40 TPP-7007 TWEAKR 61 62 63 64 65 66 67 68 69 70 TPP-2090 TWEAKR 71 72 73 74 75 76 77 78 79 80 TPP-170 Mesothelin 81 82 83 84 85 86 87 88 89 90 TPP-9476 CD123 91 92 93 94 95 96 97 98 99 100 TPP-9574 CXCR5 101 102 103 104 105 106 107 108 109 110

TPP-981, TPP-1015, TPP-2658, TPP-7007, TPP-2090, TPP-170, TPP-9476 and TPP-9574 are antibodies comprising one or more of the CDR sequences (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) given in the above table, a heavy chain (VH) variable region or a light chain (VL) variable region. Preferably, the antibody comprises a specified variable region of the heavy chain (VH) and/or a specified variable region of the light chain (VL). Preferably, the antibody comprises a specified region of the heavy chain (IgG heavy chain) and/or a specified region of the light chain (IgG light chain).

TPP-981 is an anti-EGFR antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 2, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 3, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 4, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 6, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 7, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 8.

TPP-1015 is an anti-HER2 antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 12, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 13, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 14, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 16, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 17, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 18.

TPP-2658 is an anti-TWEAKR antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 32, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 33, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 34, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 36, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 37, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 38.

TPP-7007 is an anti-TWEAKR antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 62, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 63, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 64, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 66, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 67, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 68.

TPP-2090 is an anti-TWEAKR antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 72, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 73, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 74, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 76, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 77, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 78.

TPP-170 is an anti-mesothelin antibody, comprising: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 82, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 83, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 84, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 86, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 87, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 88.

TPP-9476 is an anti-CD123 antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 92, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 93, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 94, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 96, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 97, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 98.

TPP-9574 is an anti-CXCR5 antibody, which comprises: a heavy chain (VH) variable region comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO: 102, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO: 103, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO: 104, and a light chain (VL) variable region comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO: 106, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO: 107, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 108.

TPP-981 is an anti-EGFR antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 1 and a light chain (VL) variable region represented by SEQ ID NO: 5.

TPP-1015 is an anti-HER2 antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 11 and a light chain (VL) variable region represented by SEQ ID NO: 15.

TPP-2658 is an anti-TWEAKR antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 31 and a light chain (VL) variable region represented by SEQ ID NO: 35.

TPP-7007 is an anti-TWEAKR antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 61 and a light chain (VL) variable region represented by SEQ ID NO: 65.

TPP-2090 is an anti-TWEAKR antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 71 and a light chain (VL) variable region represented by SEQ ID NO: 75.

TPP-170 is an anti-mesothelin antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 81 and a light chain (VL) variable region represented by SEQ ID NO: 85.

TPP-9476 is an anti-CD123 antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 91 and a light chain (VL) variable region represented by SEQ ID NO: 95.

TPP-9574 is an anti-CXCR5 antibody, which preferably comprises a heavy chain (VH) variable region represented by SEQ ID NO: 101 and a light chain (VL) variable region represented by SEQ ID NO: 105.

TPP-981 is an anti-EGFR antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 9 and a light chain region represented by SEQ ID NO: 10.

TPP-1015 is an anti-HER2 antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 19 and a light chain region represented by SEQ ID NO: 20.

TPP-2658 is an anti-TWEAKR antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 39 and a light chain region represented by SEQ ID NO: 40.

TPP-7007 is an anti-TWEAKR antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 69 and a light chain region represented by SEQ ID NO: 70.

TPP-2090 is an anti-TWEAKR antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 79 and a light chain region represented by SEQ ID NO: 80.

TPP-170 is an anti-mesothelin antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 89 and a light chain region represented by SEQ ID NO: 90.

TPP-9476 is an anti-CD123 antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 99 and a light chain region represented by SEQ ID NO: 100.

TPP-9574 is an anti-CXCR5 antibody, which preferably comprises a heavy chain region represented by SEQ ID NO: 109 and a light chain region represented by SEQ ID NO: 110. Abbreviations and acronyms A431NS Human tumor cell line A549 Human tumor cell line ABCBl ATP binding cassette family B member I (synonymous with P-gp and MDR1) abs. Absolute value Ac Acetyl ACN Acetonitrile ADC Antibody drug conjugate A2DC Antibody 2 drug conjugate aq. Aqueous solution ATP Adenosine triphosphate BCRP Breast cancer resistance protein, efflux transporter BEP 2-bromo-l-ethylpyridinium tetrafluoroborate Boc Tertiary butoxycarbonyl br. Broad peak (in NMR) Ex. Example BxPC3 Human tumor cell line ca. Approximately C-DAR Cysteine drug to antibody ratio (linker attached to cysteamine residue) Cl Chemical ionization (in MS) D Doublet (in NMR) D day TLC Thin layer chromatography DCI Direct chemical ionization (in MS) DCM Dichloromethane Dd Doublet Doublet (in NMR) DMAP 4-N,N-dimethylaminopyridine DME 1,2-dimethoxyethane DMEM Dulbecco's Modified Eagle Medium (standardized culture medium for cell culture) DMF N,N-dimethylformamide DMSO Dimethyl sulfoxide DAR Drug-antibody ratio DPBS, D-PBS, PBS Dulbecco's phosphate buffered saline PBS=DPBS=D-PBS, pEl 7.4, from Sigma, No. D8537. Composition: 0.2 g KCl; 0.2 g KH2PO4 (anhydrous); 8.0 g NaCl; 1.15 g Na2HPO4 (anhydrous); H2O was used to supplement II Dt Doublet Triplet (in NMR) DTT DL-dithiothreitol d. Th. Theoretical (chemical yield) EDC N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride EGFR epidermal growth factor receptor EI electron impact ionization (in MS) ELISA enzyme-linked immunosorbent assay eq. equivalent ESI electrophoresis ionization (in MS) ESI-MicroTofq ESI-MicroTofq (name of mass spectrometer, where Tof = time of flight and q = quadrupole) FCS fetal bovine serum Fmoc (9H-fluoren-9-ylmethoxy)carbonyl sat. saturated GTP guanosine-5'-triphosphate h hours HATU hexafluorophosphate O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl HCT-116 human tumor cell lineHEPES 4-(2-hydroxyethyl)piperidin-l-ethanesulfonic acidHOAc acetic acidHOAt 1-hydroxy-7-azabenzotriazoleHOBt 1-hydroxy-1H-benzotriazole hydrateHOSu N-hydroxysuccinimideHPLC high pressure high performance liquid chromatographyHT29 human tumor cell lineIC50 half inhibitory concentrationim intramuscular, administration into muscleiv intravenous, administration into veinK-DAR lysine drug to antibody ratio (linker attached to lysine residue)KPL-4 human tumor cell lineKU-19-19 human tumor cell lineLC-MS Liquid chromatography coupled mass spectrometry LLC-PK1 cells, Lewis lung carcinoma pig kidney cell line L-MDR LLC-PK1 cells transfected with human MDR1 LoVo human tumor cell line m multiplet (in NMR) Me methyl MDR1 multidrug resistance protein I MeCN acetonitrile min minutes MS mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide NCI-H292 human tumor cell line NCI-H520 human tumor cell line NMM N-methylthiophene NMP N-methyl-2-pyrrolidone NMR nuclear magnetic resonance spectroscopy NMRI mouse strain from Naval Medical Research Institute nude mice experimental animals NSCLC Non-small cell lung cancerPBS Phosphate buffered saline solutionPd/C Palladium/activated carbonP-gp P-glycoprotein, a transporterPNGaseF Enzyme used to cleave sugarsQuant Total yield (yield)quart Quartet (in NMR)quint Quintet (in NMR)RT Room temperatureRt Retention time (in HPLC)s Singlet (in NMR)sc Subcutaneous, administered under the skinSCC-4 Human tumor cell lineSCC-9 Human tumor cell lineSCID mouse, a test mouse with severe combined immunodeficiency diseaseSK-HEP-I Human tumor cell linet Triplet (in NMR)TBAF Tetra-n-butylammonium fluorideTCEP Tris(2-carboxyethyl)phosphineTEMPO (2,2,6,6-Tetramethylpiperidin-l-yl)oxytert tertiary TFA trifluoroacetic acid THF tetrahydrofuran T3P® 2,4,6-trioxide 2,4,6-tripropyl-l,3,5,2,4,6-trioxa-triphosphoracyclohexane UV ultraviolet spectrometry v/v (solution) volume ratio Z benzyloxycarbonyl 786-0 human tumor cell line amino acid abbreviation Ala = alanine Arg = arginine Asn = aspartic acid Asp = aspartic acid Cys = cysteine Glu = glutamine Gln = glutamine Gly = glycine His = histidine Ile = isoleucine Leu = leucine Lys = lysine Met = Methionine Nva = norvaline Phe = phenylalanine Pro = proline Ser = serine Thr = threonine Trp = tryptophan Tyr = tyrosine Val = valine

Depending on the linker and the coupling procedure, the ADC and A2DC shown in the structural formula of the examples (coupled to the cysteine side chain of the antibody via the cis-butylenediamide group) are present primarily in the open or closed ring form shown in each case. However, the preparation may contain small proportions of the respective other forms. The coupling reactions were carried out under argon.

The concentration of the antibody or ADC indicated in the experimental procedure can be varied without significantly affecting the results. It is also possible to use antibody or ADC concentrations in the range of 1 to 20 mg/mL in the coupling reaction, with a preferred concentration range of 5 to 15 mg/mL in the coupling reaction. It is also possible to add an excess of the precursor molecule, which may deviate from the content given in the procedure, which in turn may have an effect on the DAR. An excess of 2 to 20 equivalents of effective load precursor can be used, with a preferred range of 4 to 15 equivalents. General Procedure : Cysteine Coupling and Subsequent Ring Opening of the Thiosuccinimide Ring

Typically, a solution of 0.029 mg TCEP in 0.05 ml PBS buffer (pH 7.2) is added to 5 mg of the respective antibody (c=10 mg/ml) dissolved in 0.5 ml PBS under argon. (The concentration of the antibody solution can also vary from the standard procedure and range from 1 mg/ml to 30 mg/ml). The mixture is stirred at RT for 30 min, followed by an excess of 2 to 20 equivalents, preferably 2 to 10 equivalents, typically 7 equivalents (0.000233 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.05 mL DMSO. After stirring for another 90 min at RT, the mixture was diluted to a volume of 2.5 ml with PBS buffer pre-adjusted to pH 8, then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 8) and eluted with PBS buffer (pH 8). The solution was stirred at RT under argon at pH 8 overnight. Thereafter it was concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2) to a volume of approximately 1 to 5 mL. General procedure : Cysteine coupling

Typically, a solution of 0.029 mg TCEP in 0.05 ml PBS buffer (pH 7.2) is added to 5 mg of the respective antibody (c=10 mg/ml) dissolved in 0.5 ml PBS under argon. (The concentration of the antibody solution can also vary from the standard procedure and range from 1 mg/ml to 30 mg/ml). The mixture is stirred at RT for 30 min, followed by an excess of 2 to 20 equivalents, preferably 2 to 10 equivalents, typically 7 equivalents (0.000233 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.05 mL DMSO. After stirring for another 90 min at RT, the mixture was diluted to a volume of 2.5 ml with PBS buffer (pH 7.2), then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 7.2) and eluted with PBS buffer (pH 7.2). Thereafter, it was concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2) to a volume of approximately 1 to 5 mL. General procedure: Lysine coupling

To a solution of 5 mg of the respective antibody, typically in 0.5 mL PBS buffer (pH 7.2) (c = 10 mg/mL) (the concentration of the antibody solution can also vary from the standard procedure and range between 1 mg/ml and 30 mg/ml) under argon atmosphere, an excess of 2 to 10 equivalents, preferably 2 to 5 equivalents, typically 5 equivalents (0.00165 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.5 mL DMSO is added. After stirring at RT for 30 to 60 min, the same amount of the ADC prodriver molecule dissolved in 0.05 mL DMSO is added and stirring at RT is continued for another 30 to 60 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of about 0.300 mL by ultracentrifugation and further diluted to a volume of about 1 to 5 mL with PBS buffer (pH 7.2). ADC procatalyst molecules for the synthesis of A2DC

The following payload linker prodriver molecules are used in A2DC, and the synthesis has been described in WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578. Novel and advantageous A2DCs can be obtained from specific combinations of payloads, linkers, antibodies and/or prodriver molecules known in the art according to the formulas and examples disclosed herein. Prodriver intermediates for ADC formation of impermeable metabolites R1 LEE14057-6-1

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-propylaminoyl-N-methyl-L-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

LC-MS (method 1): R _t = 0.93 min; MS (ESIpos): m/z = 1195 [M+H] ⁺ . Intermediate R2

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-propylamino-D-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

LC-MS (method 1): R _t = R _t = 0.92 min; MS (ESIpos): m/z = 1181 [M+H] ⁺ . Intermediate R3 LEE14526-1-1

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-propylamino-N-methyl-L-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-hydroxypropyl)amino]-1-hydroxybutyl-2-yl}-L-aspartamide

LC-MS (method 1): R _t = 3.2 min; MS (ESIpos): m/z = 1177 [M+H] ⁺ . Intermediate R4 LEE14741-1-1

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-propylamino-D-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartamide

LC-MS (Method 1): R _t = R _t = 0.96 min; MS (ESIpos): m/z = 1163 [M+H] ⁺ . Intermediate R5

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-α-asparaginyl-L-asparaginyl)amino]butyryl}-β-propylamino-D-glutamine LC-MS (Method 1): R _t = R _t = 0.9 min; MS (ESIpos): m/z = 1154 [M+H] ⁺ . Intermediate R6

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(N ² -{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-asparagine)amino]butyryl}-β-propylamino-D-glutamine

LC-MS (method 1): R _t = 0.92 min; MS (ESIpos): m/z = 1039 [M+H] ⁺ . It is a driver intermediate Q1 before ADC forms permeable metabolites .

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartamide

LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 937 [M+H] ⁺ . Intermediate Q2

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-D-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartamide

LC-MS (Method 1): R _t = R _t = 1.01 min; MS (ESIpos): m/z = 937 [M+H] ⁺ . Intermediate Q3

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-propylaminoyl-L-propylaminoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartamide

LC-MS (Method 1): R _t = 1.05 min; MS (ESIpos): m/z = 919 [M+H] ⁺ . Intermediate Q4

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-propylamino-D-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartamide

LC-MS (Method 1): R _t = R _t = 1.05 min; MS (ESIpos): m/z = 919 [M+H] ⁺ . Intermediate Q5

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-D-α-asparagine-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-asparagine

LC-MS (Method 1): R _t = 0.98 min; MS (ESIpos): m/z = 981 [M+H] ⁺ . Intermediate Q6

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-α-asparagine-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-asparagine

LC-MS (Method 1): R _t = 1.03 min; MS (ESIpos): m/z = 981 [M+H] ⁺ . Intermediate Q7

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-α-aspartoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartoyl

LC-MS (Method 1): R _t = 1.04 min; MS (ESIpos): m/z = 910 [M+H] ⁺ . Intermediate Q8

N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N2-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-aspartamide

LC-MS (Method 1): Rt = 1.03 min; MS (ESIpos): m/z = 795 [M+H]+. Intermediate Q9

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-α-aspartoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-L-aspartoyl

LC-MS (Method 1): R _t = 1.07 min; MS (ESIpos): m/z = 892 [M+H] ⁺ . Effective loading of linker intermediates :

The synthesis of the payload linker prodriver molecules R1-R6 and Q1-Q9 has been described in WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578. A2DC Synthesis Part I :

For the synthesis of A2DCs illustrated in Examples I-1 to I-14, the payload linker prodrug molecules R1 to R6 and Q1 to Q9 are then coupled to the respective antibodies. The prodrug molecules can be combined with each other in different ways for the synthesis of A2DCs to achieve the desired biological profile of the respective A2DCs. Example I-1: Synthesis procedure A :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under an argon atmosphere, 4 Eq (0.16 mg) of intermediate R1 dissolved in 50 µL of DMSO was added. After stirring at RT for 30 min, 4 Eq (0.14 mg) of intermediate Q1 dissolved in 50 µL of DMSO was added, and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the following table. Synthesis Procedure A* :

To a solution of 20 mg of the respective antibody in 2 mL of PBS buffer (c = 10 mg/mL) under argon atmosphere, 4 Eq (0.63 mg) of intermediate R1 dissolved in 50 µL of DMSO were added. After stirring at RT for 30 min, 4 Eq (0.56 mg) of intermediate Q1 dissolved in 50 µL of DMSO were added and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The resulting solution was diluted to a volume of 14 mL with PBS buffer, then concentrated to 2 mL by ultracentrifugation, diluted again to a volume of 14 mL with PBS buffer (pH 7.2), and concentrated again to a final volume of 2 mL. The characteristics of the ADC batches obtained with the individual antibodies are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 1c-9476 CD123 9476 A 3.01 2.1 0.6 1.5 98.8 1e-1015 HER-2 1015 A 1.92 5.3 nd nd 100 1k-7007 TWEAKR 7007 A 2.48 3.0 1.2 1.8 94 1k-7007* TWEAKR 7007 A* 9.92 2.2 1.0 1.2 96 1m-170 Mesothelin 170 A 0.87 4.6 nd nd 98.7 1m-170* Mesothelin 170 A* 3.67 3.8 1.4 2.4 98.7 1x-9574 CXCR5 9574 A 1.02 5.4 nd nd 100 Example I-2: Synthesis procedure A :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under an argon atmosphere, 4 Eq (0.16 mg) of intermediate R2 dissolved in 50 µL of DMSO was added. After stirring at RT for 30 min, 4 Eq (0.14 mg) of intermediate Q2 dissolved in 50 µL of DMSO was added, and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the following table. Synthesis Procedure A* :

To a solution of 30 mg of the respective antibody in 2.5 mL of PBS buffer (c = 12 mg/mL) under argon atmosphere, 4 Eq (1.28 mg) of intermediate R2 dissolved in 125 µL of DMSO were added. After stirring at RT for 30 min, 4 Eq (0.77 mg) of intermediate Q2 dissolved in 125 µL of DMSO were added and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 5 mL with PBS buffer (pH 7.2), applied to 2 PD-10 columns (Sephadex® G-25 GE Healthcare), and each was eluted with 3.5 mL of PBS buffer. The resulting solution was collected and concentrated by ultracentrifugation to obtain 2 mL, diluted again with PBS buffer (pH 7.2) to a volume of 14 mL, and concentrated again to obtain a final volume of 3 mL. The characteristics of the ADC batches obtained with the individual antibodies are shown in the table below. Example I- Target mAK TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 2e-1015 HER-2 1015 A 2.03 3.1 2.1 1.0 100 2g-981 EGFR 981 A 2.19 2.3 nd nd 100 2k-7007 TWEAKR 7007 A 2.16 2.7 1.8 0.9 100 2k-7007* TWEAKR 7007 A* 8.9 3.0 1.8 1.2 100 2m-170 Mesothelin 170 A 1.83 2.5 1.6 0.9 98.1 Example I-3: Synthesis procedure B :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under argon atmosphere, 0.026 mg of TCEP dissolved in 50 µl of PBS buffer was added. After stirring at RT for 30 min, 4 Eq (0.16 mg) of intermediate R3 and 4 Eq (0.13 mg) of intermediate Q3 each dissolved in 50 µl of DMSO were added, and stirring at RT was continued for another 90 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the table below. Synthesis Procedure B* :

To a solution of 30 mg of the respective antibody in 3 mL of PBS buffer (c = 10 mg/mL) under argon atmosphere was added 0.17 mg of TCEP dissolved in 300 µl of PBS buffer. After stirring at RT for 30 min, 4 Eq (0.94 mg; 0.0008 mmol) of intermediate R3 and 4 Eq (0.74 mg) of intermediate Q3 each dissolved in 300 µl of DMSO were added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 5 mL with PBS buffer (pH 7.2), fractionated, and applied to two PD-10 columns (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL of PBS buffer each. The combined solution was then concentrated to 2 mL by ultracentrifugation and diluted again to a volume of 14 mL with PBS buffer (pH 7.2), and concentrated again to a volume of approximately 3 mL. The characteristics of the A2DC batch obtained are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 3c-9476 CD123 9476 B 2.06 3.6 nd nd 100 3e-1015 HER-2 1015 B 2.0 3.3 1.6 1.7 100 3g-981 EGFR 981 B 2.09 3.5 nd nd 100 3k-7007 TWEAKR 7007 B 2.0 2.8 1.3 1.5 100 3m-170 Mesothelin 170 B 0.51 2.6 nd nd 97.8 3x-9574* CXCR5 9574 B* 9.04 3.4 nd nd 100 Example I-4: Synthesis procedure B :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under argon atmosphere, 0.029 mg of TCEP dissolved in 50 µl of PBS buffer was added. After stirring at RT for 30 min, 4 Eq (0.16 mg) of intermediate R4 and 4 Eq (0.13 mg) of intermediate Q4 dissolved in 50 µl of DMSO were added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the following table. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 4e-1015 HER-2 1015 B 2.29 4.3 nd nd 100 4g-981 EGFR 981 B 2.1 3.8 nd nd 100 4k-7007 TWEAKR 7007 B 1.99 2.8 1.6 1.2 100 4m-170 Mesothelin 170 B 1.56 3.6 nd nd 96.7 4x-9574 CXCR5 9574 B 1.82 2.8 nd nd 100 Example I-5: Synthesis procedure A :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under argon atmosphere, 4 Eq (0.16 mg) of intermediate R2 dissolved in 50 µL of DMSO were added. After stirring at RT for 30 min, 4 Eq (0.13 mg) of intermediate Q5 dissolved in 50 µL of DMSO were added and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated to a volume of approximately 300 µL by ultracentrifugation and further diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 5c-9476 CD123 9476 A 1.95 3.0 1.6 1.4 100 5e-1015 HER-2 1015 A 1.82 5.2 4.0 1.2 97.5 5g-981 EGFR 981 A 1.93 4.1 nd nd 100 5k-7007 TWEAKR 7007 A 2.15 2.9 1.6 1.3 100 5m-170 Mesothelin 170 A 1.04 5.6 nd nd 98.2 Example I-6: Synthesis procedure A :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under an argon atmosphere, 4 Eq (0.14 mg) of intermediate Q6 dissolved in 50 µL of DMSO were added. After stirring at RT for 30 min, 4 Eq (0.16 mg) of intermediate R1 dissolved in 50 µL of DMSO were added and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated to a volume of approximately 300 µL by ultracentrifugation and further diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 6e-1015 HER-2 1015 A 1.97 3.8 2.0 1.8 100 6g-981 EGFR 981 A 1.93 4.4 nd nd 100 6k-7007 TWEAKR 7007 A 1.91 3.5 1.9 1.6 100 6m-170 Mesothelin 170 A 1.1 5.4 2.6 2.8 96.8 6x-9574 CXCR5 9574 A 1.03 4.5 2.5 2.0 100 Example I-7: Synthesis procedure A : To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under an argon atmosphere, 4 Eq (0.12 mg) of intermediate Q7 dissolved in 50 µL of DMSO were added. After stirring at RT for 30 min, 4 Eq (0.16 mg) of intermediate R5 dissolved in 50 µL of DMSO were added and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated to a volume of approximately 50 µL by ultracentrifugation and further diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 7e-1015 HER-2 1015 A 1.68 4.7 nd nd 100 7g-981 EGFR 981 A 1.86 4.1 nd nd 97.9 7k-7007 TWEAKR 7007 A 1.98 3.1 nd nd 100 7m-170 Mesothelin 170 A 1.82 3.0 nd nd 97.9 7x-9574 CXCR5 9574 A 1.58 4.6 nd nd 100 Example I-8: Synthesis procedure A :

To a solution of 5 mg of the respective antibody in 0.5 mL of PBS buffer (c = 10 mg/mL) under an argon atmosphere, 4 Eq (0.14 mg) of intermediate R6 dissolved in 50 µL of DMSO was added. After stirring at RT for 30 min, 4 Eq (0.11 mg) of intermediate Q8 dissolved in 50 µL of DMSO was added and stirring at RT was continued for another 30 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated to a volume of approximately 50 µL by ultracentrifugation and further diluted to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained ADC batch are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DAR Left DAR Right DAR Single % 8c-9476 CD123 9476 A 1.92 3.3 nd nd 100 8e-1015 HER-2 1015 A 2.05 4.3 nd Nd 89.2 8g-981 EGFR 981 A 2.19 3.5 nd nd 100 8k-7007 TWEAKR 7007 A 2.35 4.1 nd nd 86.7 8m-170 Mesothelin 170 A 2.48 4.2 1.6 2.6 95.5 Example I-11: Synthesis procedure A starting with cysteine coupling : To a solution of 10 mg of the respective antibody in 1 mL PBS buffer (c = 10 mg/mL) under argon atmosphere, 0.057 mg TCEP dissolved in 100 µl PBS buffer was added. After stirring at RT for 30 min, 7 Eq (0.55 mg) of intermediate R4 dissolved in 50 µL DMSO was added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted again to a volume of 500 µL with PBS buffer (pH 7.2). This sample was fractionated and the cysteine DAR (C-DAR) was determined, which finally also represents the C-DAR of A2DC. 250 µL of ADC intermediate was used for the subsequent lysine coupling:

250 µL of the obtained cysteine-linked ADC solution was diluted to a volume of about 550 µL with PBS buffer. Under an argon atmosphere, 10 Eq (0.31 mg) of intermediate Q2 dissolved in 50 µL DMSO was added, and the solution was stirred at RT for 2 hours. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of about 100 µL by ultracentrifugation, and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the A2DC batches obtained are shown in the table below. Synthesis procedure A* starting with cysteine coupling :

To a solution of 20 mg of the respective antibody in 1.5 mL of PBS buffer (c = 13.4 mg/mL) under argon atmosphere, 0.12 mg of TCEP dissolved in 200 µl of PBS buffer was added. After stirring at RT for 30 min, 7 Eq (1.1 mg) of intermediate R4 dissolved in 200 µL of DMSO was added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 5 mL with PBS buffer (pH 7.2), applied to two PD-10 columns (Sephadex® G-25 GE Healthcare), and eluted with x mL of PBS buffer. The obtained solutions were combined and concentrated to 200 µL by ultracentrifugation and rediluted to a volume of 2.2 mL with PBS buffer (pH 7.2). This ADC batch was used for subsequent lysine coupling and before that, the cysteine DAR (C-DAR) was determined from a small aliquot.

To a solution of 2.2 mL of the obtained cysteine-linked ADC in PBS buffer at pH 7.2 under an argon atmosphere, 10 Eq (1.23 mg) of intermediate Q2 dissolved in 200 µL of DMSO was added, and the solution was stirred at RT for 2 hours. Subsequently, the mixture was diluted to a volume of 5 mL with PBS buffer (pH 7.2). It was divided and applied to two PD-10 columns (Sephadex® G-25 GE Healthcare), and each was eluted with 3.5 mL of PBS buffer. The combined solution was then concentrated to a volume of approximately 1 mL by ultracentrifugation, then diluted again to a volume of 14 mL with PBS buffer (pH 7.2), and concentrated again to a volume of 2 mL.

The characteristics of the A2DC batch obtained are shown in the following table. Example I- Target mAb TPP- program C [mg/mL] Total DAR K-DAR C-DAR Single % 11m-170a Mesothelin 170 A 0.79 8.8 4.6 4.2 97.4 11m-170a* Mesothelin 170 A* 4.47 8.8 3.9 4.9 97.5 11k-7007a TWEAKR 7007 A 1.62 9.6 3.6 3.20 95.5 11k-2090a* TWEAKR 2090 A* 4.83 7.4 3.6 3.8 98.8 11k-2658a* TWEAKR 2658 A* 5.56 8.7 4.6 4.1 98.5 11e-1015a HER-2 1015 A 1.6 7.2 3.5 3.7 98.0 11x-9574a CXCR5 9574 A 0.54 7.3 3.0 4.3 97.7 Synthesis procedure B starting with lysine coupling

To a solution of 10 mg of the respective antibody in 1 mL of PBS buffer under argon atmosphere (c = 10 mg/mL), 5 Eq (0.31 mg) of intermediate Q2 dissolved in 50 µL of DMSO was added. After stirring at RT for 1 h, another 5 Eq (0.31 mg) of intermediate Q2 dissolved in 50 µL of DMSO was added, and stirring at RT was continued for another hour. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated to a volume of approximately 500 µL by ultracentrifugation. This sample was fractionated and the lysine DAR (K-DAR) was determined, which ultimately also represents the K-DAR of A2DC. 250 µL of this ADC intermediate was used for the subsequent cysteine coupling:

250 µL of this cysteine-linked ADC intermediate was diluted to a volume of 500 µL (c = 10 mg/mL) with PBS buffer (pH 7.2). Under an argon atmosphere, 0.029 mg TCEP dissolved in 50 µL PBS buffer was added. After stirring at RT for 30 min, 7 Eq (0.27 mg) of intermediate R4 dissolved in 50 µL DMSO was added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2) and applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with PBS buffer. The solution was concentrated to 100 µL by ultracentrifugation and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained A2DC batch are shown in the table below. Examples Target mAb TPP- program C [mg/mL] Total DRA K-DAR C-DAR Single % 11m-170b Mesothelin 170 B 0.74 5.8 1.7 4.1 97.6 11k-7007b TWEAKR 7007 B 1.53 4.8 1.7 3.1 88.9 11e-1015b HER-2 1015 B 1.23 6.2 3.3 2.8 82.3 11x-9574b CXCR5 9574 B 0.77 5.7 2.5 3.2 100 Example I-12: Synthesis procedure A starting with cysteine coupling

To a solution of 10 mg of the respective antibody in 800 µL PBS buffer (c = 12.5 mg/mL) under argon atmosphere, 0.057 mg TCEP dissolved in 100 µL PBS buffer was added. After stirring at RT for 30 min, 7 Eq (0.43 mg) of intermediate Q4 dissolved in 100 µL DMSO was added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted again to a volume of 500 µL with PBS buffer (pH 7.2). This sample was fractionated and the cysteine DAR (C-DAR) was determined, which finally also represents the C-DAR of A2DC. 250 µL of ADC intermediate was used for the subsequent lysine coupling:

250 µL of the obtained solution of cysteine-linked ADC was diluted to a volume of 550 µL with PBS buffer. Under an argon atmosphere, 10 Eq (0.4 mg) of intermediate R2 dissolved in 50 µL DMSO was added, and the solution was stirred at RT for 2 hours. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of approximately 100 µL by ultracentrifugation, and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the A2DC batch obtained are shown in the following table. Example I- Target mAb TPP- program C [mg/mL] Total DRA K-DAR C-DAR Single % 12m-170a Mesothelin 170 A 0.14 14.1 11.2 2.9 96.6 12k-7007a TWEAKR 7007 A 1.07 12.7 8.3 4.4 95.8 12e-1015a HER-2 1015 A 1.27 12.3 6.4 5.9 89.9 12x-9574a CXCR5 9574 A 1.08 8.5 5.6 2.9 95.4 Synthesis procedure B starting with lysine coupling

To a solution of 10 mg of the respective antibody in 0.9 mL of PBS buffer (c = 11.1 mg/mL) under argon atmosphere, 5 Eq (0.4 mg) of intermediate R2 dissolved in 50 µL of DMSO were added. After stirring at RT for 1 h, another 5 Eq (0.4 mg) of intermediate R2 dissolved in 50 µL of DMSO were added and stirring at RT was continued for another hour. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL of PBS buffer. The obtained solution was then concentrated to a volume of about 500 µL by ultracentrifugation. This sample was fractionated and the lysine DAR (K-DAR) was determined, which ultimately also represents the K-DAR of A2DC. 250 µL of this ADC intermediate was used for the subsequent cysteine coupling:

250 µL of this lysine-linked ADC intermediate was diluted to a volume of 500 µL (c = 10 mg/mL) with PBS buffer (pH 7.2). Under an argon atmosphere, 0.029 mg TCEP dissolved in 50 µL PBS buffer was added. After stirring at RT for 30 min, 7 Eq (0.22 mg) of intermediate Q4 dissolved in 50 µL DMSO was added and stirring was continued at RT for another 90 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2) and applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with PBS buffer. The solution was concentrated to 100 µL by ultracentrifugation and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the obtained A2DC batch are shown in the table below. Example I- Target mAb TPP- program C [mg/mL] Total DRA K-DAR C-DAR Single % 12m-170b Mesothelin 170 B 0.42 7.4 5.3 2.1 98 12k-2658b TWEAKR 2658 B 1.23 8.3 6.1 2.2 98.8 12e-1015b HER-2 1015 B 1.25 6.2 3.0 2.2 97.7 12x-9574b CXCR5 9574 B 1.19 7.0 2.2 4.8 98.5 Example I-13: Synthesis procedure:

To a solution of 10 mg of the respective antibody in 800 µL PBS buffer (c = 12.5 mg/mL) under argon atmosphere, 0.057 mg TCEP dissolved in 100 µL PBS buffer was added. After stirring at RT for 30 min, 7 Eq (0.55 mg) of intermediate R3 dissolved in 100 µL DMSO was added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted again to a volume of 500 µL with PBS buffer (pH 7.2). This sample was fractionated and the cysteine DAR (C-DAR) was determined, which finally also represents the C-DAR of A2DC. 250 µL of ADC intermediate was used for the subsequent lysine coupling:

250 µL of the obtained solution of cysteine-linked ADC was diluted to a volume of 550 µL with PBS buffer. Under an argon atmosphere, 10 Eq (0.31 mg) of intermediate Q1 dissolved in 50 µL DMSO was added, and the solution was stirred at RT for 2 hours. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of approximately 100 µL by ultracentrifugation, and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the A2DC batch obtained are shown in the following table. Example I- Target mAb TPP- program C [mg/mL] Total DRA K-DAR C-DAR Single % 13m-170 Mesothelin 170 A 0.58 8.9 4.9 4.0 95 13k-7007 TWEAKR 7007 A 1.57 8.7 5.9 2.8 78.7 13e-1015 HER-2 1015 A 0.78 11.8 7.6 4.2 86.6 13x-9574 CXCR5 9574 A 0.39 4.5 2.5 2.0 97.2 Example I-14: Synthesis procedure A :

To a solution of 10 mg of the respective antibody in 800 µL PBS buffer (c = 12.5 mg/mL) under argon atmosphere, 0.057 mg TCEP dissolved in 100 µL PBS buffer was added. After stirring at RT for 30 min, 7 Eq (0.42 mg) of intermediate Q9 dissolved in 100 µL DMSO was added and stirring was continued for another 90 min at RT. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated by ultracentrifugation and diluted again to a volume of 500 µL with PBS buffer (pH 7.2). This sample was fractionated and the cysteine DAR (C-DAR) was determined, which finally also represents the C-DAR of A2DC. 250 µL of ADC intermediate was used for the subsequent lysine coupling:

250 µL of the obtained solution of cysteine-linked ADC was diluted to a volume of 550 µL with PBS buffer. Under an argon atmosphere, 10 Eq (0.39 mg) of intermediate R5 dissolved in 50 µL DMSO was added, and the solution was stirred at RT for 2 hours. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare), and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of approximately 100 µL by ultracentrifugation, and diluted again to a volume of 2.5 mL with PBS buffer (pH 7.2). The characteristics of the A2DC batch obtained are shown in the following table. Example I- Target mAb TPP- program C [mg/mL] Total DRA K-DAR C-DAR Single % 14m-170 Mesothelin 170 A 0.79 10.5 6.8 3.7 98.5 14g-981 EGFR 981 A 1.40 7.9 4.5 3.4 98.6 14k-7007 TWEAKR 7007 A 1.83 6.9 4.0 2.9 100 14e-1015 HER-2 1015 A 1.6 8.4 5.0 3.4 98.9 14x-9574 CXCR5 9574 A 1.68 7.4 4.3 3.1 99.1 Biological Characterization CTG Analysis

Cells were grown according to standard methods using the growth medium listed under CI. The assay was performed as follows: cells were detached with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelleted, resuspended in medium, counted and seeded in 96-well culture plates with white bottom (Costar #3610) (75 pl/well, the number of cells per well was as follows: NCI-H292: 2500 cells/well, BxPC3 2500 cells/well, LoVo 3000 cells/well) and incubated in an incubator at 37°C and 5% carbon dioxide. After 24 h, 25 pi medium (fourfold concentration) containing antibody-drug conjugate was added to the cells, so that the final concentration of antibody-drug conjugate on the cells reached 3×10 ^-7 M to 3×10 ^-11 M (triplicate). The cells were then cultured in an incubator at 37°C and 5% carbon dioxide. On a parallel culture plate, at the beginning of drug treatment (day 0), cell activity was measured using the Cell Titer Glow (CTG) luminescent cell viability assay (Promega #G7573 and #G7571). For this purpose, 100 pi of substrate was added to each batch of cells, followed by covering the culture plates with aluminum foil, shaking on a plate shaker at 180 rpm for 2 minutes, standing on the bench for 8 minutes, and then measured using a luminometer (Victor X2, Perkin Elmer). The substrate detects the ATP content in living cells, generating a luminescent signal whose intensity is proportional to cell viability. After incubation with the antibody-drug conjugate for 72 h, the viability of these cells was then determined using the Cell Titer Glow luminescent cell viability assay described above. Based on the measured data, the IC50 for growth inhibition compared to day 0 was calculated using the DRC (dose response curve) analysis spreadsheet and 4-parameter fitting. The DRC analysis spreadsheet is a biobook spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).

Tables 2 and 3 below list the IC50 values of representative examples in this analysis: Table 2 : TWEAKR-A2DC Instance Number (I-) EO-PROLIF-NCI-H292 ADC CTG 72H IC50 - [mol/l] ( average) EO-Prolif-BXPC3 ADC CTG 72h IC50 - [mol/l] ( average) EO-PROLIF-LoVo ADC CTG 72H IC50 - [mol/l] ( average) 1k-7007* 1.01 E-10 1.02 E-10 ＜ 3.00 E-11 1k-7007 1.74 E-10 1.66E-10 ＜ 3.00 E-11 2k-7007* 1.27 E-10 2.15 E-10 ＜ 3.00 E-11 2k-7007 2.19 E-10 2.92 E-10 9.14 E-11 3k-7007 1.86 E-10 2.68 E-10 4.90 E-11 4k-7007 2.45E-10 3.20 E-10 6.92 E-11 5k-7007 1.04 E-10 1.67 E-10 ＜ 3.00 E-11 8k-7007 1.62 E-10 1.94 E-10 3.81 E-11 6k-7007 2.18 E-10 2.15 E-10 6.47 E-11 7k-7007 1.98 E-10 2.45E-10 7.45 E-11 11k-7007a 8.17 E-11 1.97 E-10 ＜ 3.00 E-11 11k-7007b 8.35 E-11 1.83 E-10 ＜ 3.00 E-11 12k-2658b 1.38 E-10 2.54 E-10 5.89 E-11 12k-7007a 3.04 E-10 4.95 E-10 ＜ 3.00 E-11 13k-7007 1.08 E-10 1.98 E-10 3.94 E-11 14k-7007 8.01 E-11 1.48 E-10 ＜ 3.00 E-11

The activity data reported are for the examples described in the experimental section of the present invention, where the drug/mAB ratios are indicated. The values may deviate due to different drug/mAB ratios. IC50 values are the average of several independent experiments or individual values. The effect of the antibody drug conjugates is selective for the respective isotype control comprising the respective linker and toxin group. Table 3 : CXCR5-A2DC Instance Number (I-) EO-PROLIF-Rec-1_ ADC_CTG_72h IC50 - [mol/l] ( mean value) 1x-9574 ＜ 3.00 E-11 3x-9574* 6.63 E-9 4x-9574 ＞ 3.00 E-7 6x-9574 7.26 E-9 11x-9574a 1.68 E-8 11x-9574b 1.84 E-8 13x-9574 ＜ 3.00 E-11 14x-9574 5.29 E-11 MTT assay

Cells were grown according to standard methods using the growth media listed under CI. The assay was performed as follows: Cells were detached with a solution of Accutase in PBS buffer (from Biochrom AG #L2143). Cells were pelleted and resuspended in the medium. After cell counting, a defined number of cells were plated in a 96-well culture plate with a white bottom (from Costar #3610) (NCI H292: 2500 cells/well; SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well) in a total volume of 100 µL. The cells were then incubated in an incubator at 37°C and 5% CO2 for 48 h, after which the medium was replaced with fresh medium. Antibody-drug conjugates were then added to the cells (in triplicate) at concentrations ranging from 10 ^-5 M to 10 ^-13 M in a final volume of 10 µL and incubated at 37°C and 5% CO2. After 96 h, cell proliferation was detected using the MTT assay (ATCC, Manassas, Va., USA, catalog number 30-1010K). For this purpose, MTT reagent was added to the cells followed by 4 h of incubation. Cells were lysed overnight by addition of detergent. Dye formation was detected at 570 nm using an Infinite M1000 pro, Tecan). The measured data were used to calculate the IC50 of growth inhibition using DRC (dose response curve). The proliferation of cells not treated with the test substance but treated identically in other respects was defined as a 100% value.

The activity data reported are related to the examples described in the experimental part of the present invention, where the drug/mAB ratio is indicated. The values may deviate due to different drug/mAB ratios. The IC50 values are the average of several independent experiments or individual values. The effect of the antibody drug conjugate is selective for the respective isotype control comprising the respective linker and toxin group. The results are summarized in the following tables (4, 5, 6, 7). Table 4 : Summary of mean IC50 values for mesothelin -A2DC Example (I-) Cytotoxicity assay - HT29 mesoIC50 [mol/l] 1m-170 4.75 E-09 1m-170* 1.20 E-09 2m-170 8.20 E-09 3m-170 1.26 E-09 4m-170 4.35 E-10 5m-170 6.94 E-10 8m-170 2.96 E-09 6m-170 8.49 E-10 7m-170 2.44 E-09 11m-170a 1.17 E-10 11m-170a* 1.14 E-10 11m-170b 4.75 E-10 12m-170a 1.81 E-10 12m-170b 1.27 E-10 13m-170 4.99 E-11 14m-170 3.94 E-11 Table 5 : Summary of mean IC50 values of EGFR-A2DC Example (I-) Cytotoxicity Assay - NCI H292 IC50 [mol/l] Cytotoxicity Assay - SK-Hep1 IC50 [mol/l] 2g-981 7.31 E-11 8.92 E-08 3g-981 2.59 E-11 2.97 E-09 4g-981 6.24 E-10 2.36 E-7 5g-981 1.45 E-12 1.15 E-08 8g-981 1.36 E-12 4.51 E-08 6g-981 6.20 E-12 7.20 E-10 7g-981 14g-981 Table 6 : Summary of mean IC50 values of HER2-A2DC Instance Number (I-) Cytotoxicity assay KPL4 IC50 [mol/l] 1e-1015 1.82 E-10 2e-1015 2.67E-10 3e-1015 2.17 E-10 4e-1015 3.39 E-10 5e-1015 3.17 E-10 8e-1015 2.50 E-10 6e-1015 7.88 E-11 7e-1015 2.82 E-10 11e-1015a 1.13 E-10 12e-1015a 1.73 E-10 12e-1015b 3.00 E-10 14e-1015 1.73 E-10 Table 7 : Summary of mean IC50 values of CD123-A2DC Instance Number (I-) Cytotoxicity Assay - MOLM13 IC50 [mol/l] Cytotoxicity Assay MV-4-11 IC50 [mol/l] 1c-9476 1.78 E-09 6.59 E-09 3c-9476 8.50 E-08 2.45 E-08 5c-9476 5.00 E-7 5.00 E-7 8c-9476 5.00 E-7 5.00 E-7 Internalization Analysis

Internalization is a key process that provides specific and effective cytotoxic payload by antibody drug conjugate (ADC) in cancer cells expressing antigen. This process is monitored by fluorescent labeling of specific antibodies and isotype control antibodies. First, the fluorescent dye is conjugated to the lysine of the antibody. Conjugation is performed using a two-fold molar excess of CypFier 5E mono-NFiS ester (lot 357392, GE Firecare) at pH 8.3. After coupling, the reaction mixture was purified by gel chromatography (Zeba spin desalination column, 40K, Thermo Scientific, No. 87768; elution buffer: DULBECCO'S PBS, Sigma-Aldrich, No. D8537) to eliminate excess dye and adjust the pH. The protein solution was concentrated using a VIVASPIN 500 column (Sartorius stedim biotec). The dye loading of the antibody was determined by spectrophotometric analysis (from NanoDrop) and subsequent calculation (D/P = A _dye e _protein : (A ₂₈₀ -0.16A _dye ) e _dye ).

Dye loading was of comparable magnitude for the antibodies and isotype controls tested here. In cell binding assays, it was demonstrated that conjugation did not cause a change in antibody affinity.

Labeled antibodies were used for internalization analysis. Before starting treatment, cells (2×10 ⁴ /well) were seeded in 96-well MTP (coarse, black, clear bottom, No. 4308776, from Applied Biosystems) in 100 µl of medium. After incubation for 18 h at 37°C/5% CO ₂ , the medium was replaced and labeled antibodies were added at different concentrations (10, 5, 2.5, 1, 0.1 pg/ml). The same treatment scheme was applied to the labeled isotype control (negative control). The selected incubation times were 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6 h and 24 h. Fluorescence measurements were performed using InCe- IlAnalyzer 1000 (from GE Healthcare). Kinetic evaluation was then performed by measuring the parameters particle count/cell and total particle density/cell. After binding to the receptor, the internalization capacity of the antibodies was tested. For this purpose , cells with different receptor expression levels were selected. Target-mediated specific internalization of the antibodies was observed, while the isotype control showed no internalization. Quantification of metabolites formed by A2DC

To quantify A2DC metabolites released from A2DC, cancer cells were incubated with A2DC from Example 6 for 72 h. Cell lysates and corresponding supernatants were collected at each time point, and active payload metabolites were quantified by LC-MS. For calibration, 0.6 to 1000 µg/L active metabolite 8 was added to the cell homogenate (for cell lysates) or cell culture medium (for supernatants). Samples were analyzed using HPLC coupled to a triple quadrupole mass spectrometer API 4500 (AB Sciex). In vivo activity test Growth inhibition / regression of experimental tumors in mice

Human (NCI-H292) or mouse (CT26, MC38) tumor cells expressing the antigen of the antibody drug conjugate are inoculated subcutaneously into the flank of immunosuppressed mice (e.g., NMRi nude mice or SCID mice) or immunocompetent mice (e.g., Balb/c or C57Bl/6 mice). One million to ten million cells are isolated from the cell culture, centrifuged and resuspended in culture medium or culture medium/Matrigel. The cell suspension is injected subcutaneously into the mouse. Within a few days, tumors grow. Treatment is initiated after tumors have established and are approximately 40 ^mm2 in size. To test the effect on larger tumors, treatment may be initiated when the tumor size is 50-100 ^mm2 . A2DC treatment was performed intraperitoneally or via the intravenous (iv) route into the tail vein of mice. A2DC was administered in a volume of 5 ml/kg.

The treatment regimen depends on the pharmacokinetics of the antibody. For the conjugates according to the invention, as a standard, treatments are carried out once or twice a week for 2 or 3 weeks. For highly effective compounds, a regimen with a single treatment can be adopted. However, the treatment can also be continued, or a second cycle with three treatment days can be continued later. As a standard, 8 animals are used per treatment group. In addition to the group to which the active substance is administered, one group is treated only with buffer according to the same regimen as a control group. During the experiment, the two-dimensional area (length/width) of the tumor is measured regularly using a caliper. The tumor area is measured as length×width. The ratio of the mean tumor area of the treatment group to the mean tumor area of the control group was expressed as T/C _area .

A2DC example I-1k-7007* was tested for binding to the TWEAK receptor at a dose of 5 mg/kg in mice bearing highly antigen-expressing NCI-H292 tumors. Treatments were performed once a week for three times, and example I-1k-7007* was compared to the homotypic conjugate and vehicle (PBS). Potent tumor suppression was observed for example I-1k-7007* starting shortly after the first dose, which lasted for approximately 40 days, after which tumors regrew. The homotypic conjugate showed only a slight effect compared to the vehicle. ( Figure 3 )

In addition to this xenograft model, syngeneic (mouse) tumor models CT26 and MC38 were also studied, both of which express TWEAK receptors as confirmed by immunohistochemistry. A2DC Example I 11k-2658* was injected intraperitoneally into CT26 tumor-bearing mice (Balb/c, female) at two different doses (2.5 and 5 mg/kg) in a single treatment, and tumor growth was observed to last for about 3 weeks. Therefore, a stronger dose-dependent tumor growth inhibition of Example I-11k-2658* was demonstrated. A similarly stronger efficacy of 11k-2658* was found in MC38 tumor-bearing mice treated three times with 5 mg/kg. ( Figure 4 )

In all studies, the A2DC compound was well tolerated and did not affect the body weight or behavior of the rodents. No suspicious findings were found at gross necropsy.

A summary of the T/C _area ratios from studies in different tumor models is listed below in Table 8. Table 8 : Summary of T/C area ratios from studies in different tumor models A2DC Instance (I-) Tumor Model Dosage T/C _area ( days) 1k-same type NCI-H292 5 mg/kg, q7dx3 0.65 (d22) 1k-7007* NCI-H292 5 mg/kg, q7dx3 0.09 (d22) 11k-2658* CT26 2.5 mg/kg, SD 0.30 (d17) 11k-2658* CT26 5 mg/kg, SD 0.15 (d17) 11k-2658* MC38 5 mg/kg, q3dx3 0.26 (d18) SD: Single Dose Part II: Preparation and Characterization of Compounds of Formula II and III and Synthetic Antibody -2- Drug Conjugates (A2DC)

Antibodies suitable for use in Part II of the present invention include but are not limited to those disclosed in Table 1 of Part I. General Methods for Expressing Antibodies in Mammalian Cells

Antibodies such as TPP-2658, TPP-2090, TPP-7007, TPP-1015, TPP-170, TPP-6013, TPP-9476, TPP-9574, and TPP-981 are produced in transient mammalian cell culture as described in Tom et al., Chapter 12 of Methods Express: Expression Systems , ed. Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. General Methods for Purifying Antibodies from Cell Supernatants

Antibodies, such as TPP-2658, TPP-2090, TPP-7007, TPP-1015, TPP-170, TPP-6013, TPP-9476, TPP-9574 and TPP-981, were obtained from cell culture supernatants. The cell supernatants were clarified by centrifugation of the cells. The cell supernatants were then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. For this purpose, the column was equilibrated in DPBS (pH 7.4) (Sigma/Aldrich), the cell supernatant was applied, and the column was washed with approximately 10 column volumes of DPBS (pH 7.4) + 500 mM sodium chloride. Antibodies were eluted in 50 mM sodium acetate (pH 3.5) + 500 mM sodium chloride and further purified by gel filtration on a Superdex 200 column (GE Healthcare) in DPBS (pH 7.4).

Commercial antibodies were purified from commercial products by standard chromatographic methods (protein A chromatography, preparative gel filtration chromatography (SEC-size exclusion chromatography)). General procedure for cysteine coupling

The following antibodies were used in the exemplary coupling schemes described in the A2DC Examples section: Example a: Anti-EGFR mAb (cetuximab) (TPP981) Example e: Anti-Her2 mAb (TPP1015) Example k: Anti-TWEAKR mAb (TPP-7007) Example k: Anti-TWEAKR mAb (TPP-2658) Example k: Anti-TWEAKR mAb (TPP-2090) Example c: Anti-CD123 mAb (TPP-9476) Example x: Anti-CXCR5 mAb (TPP-9574)

To obtain the thiol nucleophile, the antibody can be reduced by the state of the art method also employed in the examples described below. Alternatively, free cysteine residues can be site-specifically engineered into the antibody. Coupling reactions with the electrophiles described in the section "ADC Prodriver Molecules" are typically performed under argon as described in the general procedure in the section "A2DC Examples".

In the bioassay, the concentration of the final A2DC sample was adjusted to the range of 0.5 to 15 mg/ml by re-dilution as necessary. The respective protein concentrations in the A2DC solutions described in the examples were determined. In addition, the antibody loading (drug/mAb ratio) was determined using the methods described herein.

Depending on the linker, the A2DC shown in the examples may also be present in lower or higher levels in the hydrolyzed succinylamide form linked to the antibody.

Specifically, by connecting sub-substructures KSP-I-A2DC linked to the thiol group of the antibody can optionally also be hydrolyzed after coupling by rebuffering and stirring at pH 8 for about 20 to 24 h to obtain A2DC linked via the open chain succinamide.

#1 indicates the sulfide bridge to the antibody, and #2 indicates the point of attachment to the modified KSP inhibitor.

Such linkers, A2DC linked to the antibody via hydrolyzed unsuccinamide, show higher stability against deconjugation by reverse Michael reaction and can be prepared as shown in the following scheme and described in General Procedure A (Lerchen et al., Angew. Chem. Int. Ed. 2018, 57, 15243):

Other potentially hydrolysis-sensitive thianylsuccinamide bridges to the antibody may contain the following linker substructures, where #1 represents the thioether bond to the antibody and #2 represents the point of attachment to the modified KSP inhibitor:

In the structural formula described in the examples, AK ₁ may mean: Example a: anti-EGFR mAb (cetuximab) (TPP981), partially reduced Example e: anti-Her2 mAb (TPP1015), partially reduced Example k-7007: anti-TWEAKR mAb (TPP-7007), partially reduced Example k-2658: anti-TWEAKR mAb (TPP-2658), partially reduced Example k-2090: anti-TWEAKR mAb (TPP-2090), partially reduced Example c: anti-CD123 mAb (TPP-9476), partially reduced Example x: anti-CXCR5 mAb (TPP-9574), partially reduced wherein § ¹ represents the bond to the succinimidyl group or to any isomeric hydrolytically releasable succinimidyl group or the alkylene group derived therefrom, and S S represents the sulfur atom of the cysteine residue of the partially reduced antibody. General Procedure for Lysine Coupling

The following antibodies were used in the exemplary coupling schemes described in the A2DC Examples section: Example a: Anti-EGFR mAb (cetuximab) (TPP981) Example e: Anti-Her2 mAb (TPP1015) Example k: Anti-TWEAKR mAb (TPP-7007) Example k: Anti-TWEAKR mAb (TPP-2658) Example k: Anti-TWEAKR mAb (TPP-2090) Example c: Anti-CD123 mAb (TPP-9476) Example x: Anti-CXCR5 mAb (TPP-9574)

In the structural formula described in the examples, AK ₂ may refer to: Example a: Anti-EGFR mAb (cetuximab) (TPP981) Example e: Anti-Her2 mAb (TPP1015) Example k-7007: Anti-TWEAKR mAb (TPP-7007) Example k-2658: Anti-TWEAKR mAb (TPP-2658)

Example k-2090: Anti-TWEAKR mAb (TPP-2090) Example c: Anti-CD123 mAb (TPP-9476) Example x: Anti-CXCR5 mAb (TPP-9574) Where § ² represents the bond to the carbonyl group and NH represents the pendant amine group in the lysine residue of the antibody. Analytical characterization of A2DC Drug loading determination and purity assessment by SEC-UV

Size exclusion chromatography (SEC) was used to determine the drug-antibody ratio (DAR) and purity of each A2DC. 50 μL of A2DC solution was used for analysis. SEC analysis was performed on a 1200 HPLC system monitored at 260 nm and 280 nm. A Superdex 200 10/300 GL column (GE Healthcare) was operated at a flow rate of 0.5 mL/min using phosphate buffered saline (PBS) with an isocratic gradient at room temperature. The dimer and aggregate content was determined from the integration of the UV peaks of monomers, dimers, and aggregates. To calculate the DAR, the area under the SEC peak curve at 260 nm (A _drug ) and 280 nm (A ₂₈₀ ) (the sum of aggregates, dimers, and monomers) was calculated using the following equation: (Ab = antibody, D = drug, ldrug = wavelength of interest for the drug (260 nm for the KSP toxin group), ε = absorption coefficient, A = absorption at a specific wavelength)

For toxin boluses, experimentally determined absorption coefficients are used. For all antibodies, a set of common absorption coefficients is used (see table below). Absorption coefficients of antibodies and drugs . Wavelength(nm) antibody KSP 280 ε _Ab ²⁸⁰ 0.2284 ε _drug ²⁸⁰ 0.010 260 ε _Ab ²⁶⁰ 0.1163 ε _drug ²⁶⁰ 0.014 Determination of A2DC concentration

The concentration of each A2DC ( _CA2DC ) was determined by measuring the absorption at 280 nm. The concentration was calculated using the absorption coefficient of the respective antibody. The concentration was corrected to take into account the absorbance of the toxin bolus at 280 nm using the following equation: _CA2DC = initial concentration / (1 + DAR _UV * [εdrug _{280 nm} / _{εAntibody 280 nm} ]) where initial concentration = concentration calculated using only the extinction coefficient of the antibody, DAR _UV = drug loading of the respective A2DC determined by UV absorption, εdrug _{280 nm} = extinction coefficient of drug at 280 nm, and εAntibody _{280 nm} = extinction coefficient of antibody at 280 nm). Characterization of A2DC by mass spectrometry

As an alternative to characterization of antibodies and toxigenic species and for DAR determination, ADC and A2DC have been characterized by mass spectrometry.

Molecular weight analysis was performed using a combination of HPLC and ESI-Q-TOF consisting of an I-class HPLC (Waters) for sample desalting and separation, and an Impact HD mass spectrometer (Bruker Daltonik, Bremen) equipped with instrument control and acquisition software HyStar 3.2, ESI Compass 1.7, and Maximum Entropy Deconvolution Option for MS analysis.

Chromatographic system and conditions : Column: Acquity UPLC BEH300C4 1.7 µm, 1.0×50 mm; Column temperature: 70°C; Flow rate: 200 µl/min; Mobile phase solution A: 0.1% formic acid, 94.9% water, 5% ACN; Mobile phase solution B: 0.1% formic acid, 9.9% water, 10% ACN and 80% 2-propanol.

Binary gradient profile of intact antibody : 2min 5% B, 2.5min 50% B, 3.5min 50% B, 5min 95% B, 5.1min 5% B, 5.6min 95% B, 5.7min 5% B, 6.2min 95% B, 6.3min 5% B, 7.5min 5% B

Binary gradient profile of reduced antibody : 2 min 5% B, 4 min 30% B, 5 min 50% B, 7.5 min 50% B, 8.5 min 95% B, 8.6 min 5% B, 9.1 min 95% B, 9.3 min 5% B, 9.8 min 95% B, 9.9 min 5% B, 12 min 5% B

For Cys- coupled A2DC , the determination of the molecular weight of the individual conjugate species has been measured after deglycosylation and reduction. Dilute about 160 pmol of conjugate with 20 mM sodium phosphate (pH 6.5) in a final volume of 25 µl. Add 1 µl PNGase F and incubate the sample overnight at 37°C with gentle shaking (Thermomixer). Denature 10 µl of the deglycosylated sample at 60°C by 20 µl 5M Gu*HCl (guanidine hydrochloride in 50 mM triethylammonium bicarbonate) and boil for about 5 min. To reduce the protein, add 0.5 µl of 255 mM DTT dissolved in water and incubate the mixture at 60°C for about 10 min. The cooled samples were acidified with 2 µl of 10% formic acid/water and then analyzed by mass spectrometry as described above. For DAR determination, all spectra relative to the signal in the TIC (total ion chromatogram) were summed and the molecular weights of the different conjugate species were calculated based on MaxEnt decyclization of the light and heavy chains. The average loading of antibodies with toxic groups was calculated based on the peak area determined by integrating twice the sum of the HC loading and LC loading, where the HC loading was the sum of the weighted integration results of the number of toxic groups of all heavy chain (HC) peaks divided by the sum of the individual weighted integration results of the HC peaks, and the LC loading was the sum of the weighted integration results of the number of toxic groups of the light chain (LC) peaks divided by the sum of the individual weighted integration results of all LC peaks.

For Lys- coupled A2DC , the determination of the molecular weight of the individual conjugate species has been measured after deglycosylation. Dilute approximately 160 pmol of conjugate with 20 mM sodium phosphate (pH 6.5) in a final volume of 25 µl. Add 1 µl PNGase F and incubate the sample overnight at 37°C with gentle shaking (Thermomixer). Acidify 5 µl of the deglycosylated sample with 2 µl of 10% formic acid/water and then dilute to a concentration of 1 pmol/µl with 0.1% formic acid/water. Analyze 3 µl by mass spectrometry as described above. For DAR determination, all spectra relative to the signal in the TIC (total ion chromatogram) were summed and the molecular weights of the different conjugate species were calculated based on MaxEnt decyclization of intact antibodies with different numbers of toxin groups. DAR was calculated by summing the peak areas weighted for the number of toxin groups and dividing by the sum of the unweighted peak areas. Examination of antigen binding of A2DC

After coupling has taken place, the ability to bind to the target molecule is checked. Those skilled in the art are familiar with a variety of methods that can be used for this purpose; for example, the affinity of the conjugate can be checked using ELISA techniques or surface plasmon resonance analysis (BIA-core™ measurement). Those skilled in the art can measure the concentration of the conjugate using customary methods, such as by protein assays for antibody conjugates (see also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Poison et al., Blood 2007; 1102:616-623). Analytical methods (LC-MS)

Method 1 (LC-MS): Instrument: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8 µ 50×1 mm; Mobile phase A: 1 L water + 0.25 mL 99% formic acid, Mobile phase B: 1 L acetonitrile + 0.25 mL 99% formic acid; Gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; Oven: 50°C; Flow rate: 0.40 ml/min; UV detection: 208-400 nm.

Method 2 (LC-MS): System MS: Thermo Scientific FT-MS; System UHPLC+: Thermo Scientific UltiMate 3000; Column: Waters, HSST3, 2.1×75 mm, C18 1.8 µm; Solvent A: 1 l water + 0.01% formic acid; Solvent B: 1 l acetonitrile + 0.01% formic acid; Gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; Oven: 50°C; Flow rate: 0.90 ml/min; UV-detection: 210 nm/ Optimum Integration Path 210-300 nm

Method 3 (LC-MS): Instrument: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8 µm 50×1 mm; Solvent A: 1 l water + 0.25 ml 99% IGE formic acid, Solvent B: 1 l acetonitrile + 0.25 ml 99% IGE formic acid; Gradient: 0.0 min 95% A → 6.0 min 5% A → 7.5 min 5% A, Oven: 50°C; Flow rate: 0.35 ml/min; UV detection: 210 nm.

Method 4 (LC-MS): System MS: ThermoFisherScientific LTQ-Orbitrap-XL; System HPLC: Agilent 1200SL; Column: Agilent, POROSHELL 120, 3×150 mm, SB - C18 2.7 µm; Solvent A: 1 l water + 0.1% trifluoroacetic acid; Solvent B: 1 l acetonitrile + 0.1% trifluoroacetic acid; Gradient: 0.0 min 2% B → 0.3 min 2% B → 5.0 min 95% B → 10.0 min 95% B; Oven: 40°C; Flow rate: 0.75 ml/min; UV detection: 210 nm

Method 5 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters, HSST3, 2.1×50 mm, C18 1.8 µm; Solvent A: 1 l water + 0.01% formic acid; Solvent B: 1 l acetonitrile + 0.01% formic acid; Gradient: 0.0 min 2% B → 0.5 min 2% B → 7.5 min 95% B → 10.0 min 95% B; Oven: 50°C; Flow rate: 1.00 ml/min; UV detection: 210 nm

Method 6 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters Acquity UPLC HSS T3 1.8 µm 50×1 mm; Solvent A: 1 l water + 0.100 ml 99% IGE formic acid, Solvent B: 1 l acetonitrile + 0.100 ml 99% IGE formic acid; Gradient: 0.0 min 90% A →1.2 min 5% A →2.0 min 5% A Oven: 50°C; Flow rate: 0.40 ml/min; UV detection: 210 nm. Linker intermediate Intermediate 1

N-[(Benzyloxy)carbonyl]-L-alanine yl-N-methyl-L-alanine tributyl ester

To a solution of N-[(benzyloxy)carbonyl]-L-alanine (750 mg, 3.36 mmol) in DMF (25 mL) was added N-methyl-L-alanine tributyl ester hydrochloride (723 mg, 3.70 mmol), N,N-diisopropylethylamine (1.6 mL, 9.1 mmol) and HATU (1.66 g, 4.37 mmol), and the reaction was stirred at room temperature for 30 minutes. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 1.14 g of the product. LC-MS (Method 1): Rt = 1.01 min; MS (ESIpos): m/z = 364 (M+H) ⁺ intermediate 2

N-[(Benzyloxy)carbonyl]-L-alanine yl-N-methyl-L-alanine

Tert-butyl-N-[(benzyloxy)carbonyl]-L-propylaminoyl-N-methyl-L-alaninate (1.14 g, 3.11 mmol) was diluted in dichloromethane (20 mL). Trifluoroacetic acid (20 mL) was added and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure and the residue was diluted in acetonitrile/water and lyophilized to give 1.16 g of product. LC-MS (Method 1): Rt = 0.68 min; MS (ESIpos): m/z = 308 (M+H) ⁺ . Intermediate 3

N-[(Benzyloxy)carbonyl]-L-propylaminoyl-N-methyl-L-propylaminoyl-L-aspartate tributyl ester

To a solution of N-[(benzyloxy)carbonyl]-L-propylaminoyl-N-methyl-L-alanine (592 mg, 1.92 mmol) in DMF (32 mL) was added tributyl L-aspartate (398 mg, 2.11 mmol), N,N-diisopropylethylamine (1.0 mL, 5.8 mmol) and HATU (949 mg, 2.50 mmol), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 898 mg of the product. LC-MS (Method 1): Rt = 0.75 min; MS (ESIpos): m/z = 478 (M+H) ⁺ . Intermediate 4

N-[(Benzyloxy)carbonyl]-L-propylaminoyl-N-methyl-L-propylaminoyl-L-aspartic acid

Tert-butyl-N-[(benzyloxy)carbonyl]-L-propylamino-N-methyl-L-propylamino-L-aspartate (2.81 g, 5.88 mmol) was diluted in dichloromethane (66 mL). Trifluoroacetic acid (33 mL) was added and the reaction was stirred at room temperature for 3 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 848 mg of product. LC-MS (Method 1): Rt = 0.57 min; MS (ESIpos): m/z = 422 (M+H) ⁺ Intermediate 5

N-[(Benzyloxy)carbonyl]-L-propylaminoyl-L-propylaminoyl-L-aspartic acid

The title compound was prepared as described in WO2017/162663. Intermediate 6

L-Alanyl-L-Alanyl-L-Aspartic Acid

The title compound was prepared as described in WO2017/162663. Intermediate 7

N-{[2-(Trimethylsilyl)ethoxy]carbonyl}-L-propylaminoyl-L-propylaminoyl-L-aspartic acid

To a solution of L-propylamino-L-propylamino-L-aspartic acid (270 mg, 984 µmol) in DMF (20 ml) were added 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (306 mg, 1.18 mmol) and N , N -diisopropylethylamine (210 µl, 1.2 mmol]), and the reaction was stirred at room temperature for 4 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 247 mg of the product. LC-MS (Method 1): R _t = 0.77 min; MS (ESIpos): m/z = 419 (M+H) ⁺ Intermediate 8

N-[(Benzyloxy)carbonyl]-L-propylaminoyl-L-aspartic acid 4-benzyl 1-tert-butyl ester

To a solution of N-[(benzyloxy)carbonyl]-L-alanine 2,5-dioxopyrrolidin-1-yl ester (719 mg, 2.24 mmol; CAS3401-36-3) and L-aspartic acid 4-benzyl 1-tert-butyl ester (700 mg, 2.04 mmol) in DMF (9 mL) was added N , N -diisopropylethylamine (1.2 ml, 7.1 mmol). The reaction was stirred at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 988 mg of the product. LC-MS (Method 1): R _t = 1.11 min; MS (ESIpos): m/z = 485 (M+H) ⁺ intermediate 9

(2S)-4-(Benzyloxy)-2-{[(2S)-2-{[(Benzyloxy)carbonyl]amino}propionyl]amino}-4-hydroxybutyric acid

To a solution of N-[(benzyloxy)carbonyl]-L-propylaminoyl-L-aspartic acid 4-benzyl 1-t-butyl ester (988 mg, 1.96 mmol) in dichloromethane (2.0 ml) was added trifluoroacetic acid (2.0 ml) and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure to give 870 mg of the product. LC-MS (Method 1): R _t = 0.86 min; MS (ESIpos): m/z = 429 (M+H) ⁺ intermediate 10

(5S,8S,11S)-11-(2-amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecane-12-oic acid tributyl ester

N , N -diisopropylethylamine (1.9 ml, 11 mmol; CAS-RN: [7087-68-5]) was slowly added to a solution of (2S)-4-(benzyloxy)-2-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-4-oxobutanoic acid (988 mg, 2.21 mmol), L-aspartic acid tributyl ester (511 mg, 2.65 mmol) and HATU (1.01 g, 2.65 mmol; CAS-RN: 148893-10-1) in DMF (10 ml), and the reaction was stirred at room temperature for 5 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 988 mg of product. LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 599 (M+H) ⁺ intermediate 11

(5S,8S,11S)-11-(2-amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxo-4,7,10-triazadodecane-12-oic acid

(5S,8S,11S)-11-(2-amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecane-12-oic acid tributyl ester (989 mg, 1.65 mmol) was dissolved in a mixture of dichloromethane (5.0 ml) and trifluoroacetic acid (5.0 ml), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was diluted in diethyl ether. The solid was filtered off to obtain 850 mg of the title product. LC-MS (method 1): R _t = 0.76 min; MS (ESIpos): m/z = 543 (M+H) ⁺ intermediate 12

(2S)-2-{[(2S)-2-{[(benzyloxy)carbonyl]amino}-5-tributyloxy-5-oxopentanyI]amino}pentanedioic acid 1-benzyl 5-tributyl ester

To a solution of L-glutamine 1-benzyl 5-tert-butyl ester hydrochloride (100 mg, 303 µmol) and (2S)-2-{[(benzyloxy)carbonyl]amino}-5-tert-butyloxy-5-oxopentanoic acid (102 mg, 303 µmol) in DMF (10 ml) was added HATU (138 mg, 364 µmol; CAS-RN: 148893-10-1) and N,N -diisopropylethylamine (160 µl, 910 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 5 min. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 166 mg of the product. LC-MS (Method 2): R _t = 2.44 min; MS (ESIpos): m/z = 613 (M+H) ⁺ intermediate 13

(4S)-4-{[(Benzyloxy)carbonyl]amino}-5-{[(2S)-1-(Benzyloxy)-4-carboxy-1-oxobutyl-2-yl]amino}-5-oxopentanoic acid

(2S)-2-{[(2S)-2-{[(benzyloxy)carbonyl]amino}-5-tributyloxy-5-oxopentanyl]amino}pentanedioic acid 1-benzyl ester 5-tributyl ester (166 mg, 271 µmol) was dissolved in a mixture of trifluoroacetic acid (5.0 ml) and dichloromethane (10 ml), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was diluted in acetonitrile/water and lyophilized to give 134 mg of the product. LC-MS (Method 2): Rt = 1.44 min; MS (ESIpos): m/z = 501 (M+H) ⁺ intermediate 14

N-(tert-butyloxycarbonyl)-L-propylaminoyl-N-methyl-L-alanine

To a solution of N-methyl-L-alanine (500 mg, 4.85 mmol) in DMF (50 ml) were added N-(tert-butyloxycarbonyl)-L-alanine 2,5-dioxopyrrolidin-1-yl ester (1.53 g, 5.33 mmol) and N , N -diisopropylethylamine (1.7 ml, 9.7 mmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 14 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 2.1 g of the product. LC-MS (Method 1): R _t = 0.63 min; MS (ESIpos): m/z = 275 (M+H) ⁺ intermediate 15

N-[(Benzyloxy)carbonyl]-β-propylaminoyl-D-glutamine di-tributyl ester

To a solution of N-[(benzyloxy)carbonyl]-β-alanine (755 mg, 3.38 mmol) and D-glutamine di-tert-butyl ester-hydrochloric acid (1.00 g, 3.38 mmol) in DMF (20 ml) were added HATU (1.67 g, 4.39 mmol; CAS-148893-10-1) and N,N-diisopropylethylamine (1.2 ml, 6.8 mmol; CAS-7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure, and the residue was dissolved in dichloromethane and water. The organic phase was concentrated, and the residue was purified by preparative HPLC to give 1.4 g of the product. LC-MS (method 1): R _t = 1.07 min; MS (ESIpos): m/z = 465 (M+H) ⁺ intermediate 16

β-Alanyl-D-glutamine di-tert-butyl ester

To a solution of N-[(benzyloxy)carbonyl]-β-propylaminoyl-D-glutamine di-tert-butyl ester (1.44 g, 3.10 mmol) in methanol/DCM (200 ml, 1:1) was added 10% palladium/activated carbon (100 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was lyophilized to give 1.01 g of the title product. LC-MS (Method 1): R _t = 0.61 min; MS (ESIpos): m/z = 331 (M+H) ⁺ intermediate 17

N-(Bromoacetyl)-β-propylaminoyl-D-glutamine di-tert-butyl ester

To a solution of β-propylaminoyl-D-glutamine di-tert-butyl ester (80.7 mg, 225 µmol) and bromoacetic anhydride (61.3 mg, 236 µmol) in dichloromethane (9.8 ml) was added N , N -diisopropylethylamine (120 µl, 670 µmol) and the reaction was stirred at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative column chromatography to give 70 mg of the product. LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 451 (M+H) ⁺ intermediate 18

N-(Bromoacetyl)-β-propylaminoyl-D-glutamine

N-(Bromoacetyl)-β-propylaminoyl-D-glutamine di-tert-butyl ester (69.5 mg, 154 µmol) was dissolved in a mixture of DCM/TFA (2 ml, 1:1) and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure and the residue was diluted in acetonitrile/water and lyophilized. The crude product was purified by preparative HPLC to give 45 mg of the product. LC-MS (Method 1): R _t = 0.19 min; MS (ESIpos): m/z = 339 (M+H) ⁺ intermediate 19

N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-propylaminoyl-N-methyl-L-alanine tributyl ester

To a solution of N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-alanine (1.92 g, 6.16 mmol) and N-methyl-L-alanine tributyl ester-hydrochloric acid (1.33 g, 6.78 mmol) in DMF (30 ml), HATU (3.05 g, 8.01 mmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (2.9 ml, 17 mmol; CAS-RN: 7087-68-5) were added, and the reaction was stirred at room temperature for 15 min. A mixture of water (5 mL) and 0.1% TFA was added, and the reaction was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 2.6 g of the product. LC-MS (method 1): R _t = 1.23 min; MS (ESIpos): m/z = 453 [M+H] ⁺ . ¹H-NMR (400 MHz, DMSO-d6) δ [ppm]: -0.008 (0.59), 0.008 (0.45), 1.175 (0.50), 1.190 (2.41), 1.207 (2.21), 1.229 (2.37), 1.247 (2.34), 1.32 0 (0.41), 1.338 (0.50), 1.354 (2.74), 1.374 (16.00), 2.681 (0.79), 2.914 (4.60), 4.200 (0.60), 4.216 (0.69), 4.247 (1.71), 4.262 (0.76 ), 4.491 (0.51), 4.820 (0.60), 4.839 (0.61), 7.306 (0.73), 7.325 (1.76), 7.343 (1.15), 7.415 (1.97), 7.434 (0.88), 7.550 (0.56), 7.569 (0.55), 7.699 (0.71), 7.717 (1.25), 7.734 (0.67), 7.880 (2.10), 7.898 (1.96). Intermediate 20

N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-propylaminoyl-N-methyl-L-alanine

N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-propylaminoyl-N-methyl-L-alanine tributyl ester (2.63 g, 5.81 mmol) was dissolved in dichloromethane (25 ml) and TFA (9.0 ml, 120 mmol; CAS-RN: 76-05-1) was added. The reaction was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure and dried under high vacuum to give 2.9 g of crude product, which was used in the subsequent step without further purification. LC-MS (Method 1): R _t = 0.90 min; MS (ESIpos): m/z = 397 [M+H] ⁺ . ¹H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.017 (0.52), 1.036 (0.52), 1.110 (0.45), 1.179 (9.09), 1.197 (8.91), 1.262 (8.31), 1.280 (8.15), 1.35 8 (2.00), 1.375 (2.23), 2.328 (0.44), 2.366 (0.40), 2.523 (1.62), 2.670 (1.28), 2.689 (0.62), 2.709 (3.41), 2.731 (0.89), 2.890 (1.19) , 2.909 (16.00), 4.184 (1.02), 4.200 (2.62), 4.218 (3.17), 4.253 (5.43), 4.268 (2.48), 4.274 (2.25), 4.455 (0.43), 4.473 (1.29), 4.491 (1.86), 4.509 (1.29), 4.528 (0.44), 4.927 (0.76), 4.945 (2.18), 4.963 (2.13), 4.981 (0.68), 7.307 (3.01), 7.325 (7.17), 7.344 (4.71), 7.396 (4.68), 7.415 (7.65), 7.433 (3.40), 7.557 (1.94), 7.576 (1.91), 7.702 (3.07), 7.720 (5.18), 7.738 (2.46), 7.879 (8.13), 7.898 (7.54). Intermediate 21

N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-propylaminoyl-N-methyl-L-propylaminoyl-L-aspartic acid tributyl ester

To a solution of N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-propylaminoyl-N-methyl-L-alanine (2.30 g, 5.81 mmol) and tributyl L-aspartate (1.20 g, 6.39 mmol) in DMF (25 ml) were added HATU (2.87 g, 7.55 mmol; CAS-RN: [148893-10-1]) and N,N-diisopropylethylamine (2.7 ml, 16 mmol; CAS-RN: [7087-68-5]), and the reaction was stirred at room temperature for 15 min. The reaction was quenched by adding water (5 mL) and 0.1% TFA, and the mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 3.4 g of product. LC-MS (Method 2): R _t = 1.83 min; MS (ESIpos): m/z = 567 [M+H] ⁺ . ¹H-NMR (400 MHz, DMSO-d6) δ [ppm]: -0.008 (2.08), 0.008 (1.62), 1.193 (2.42), 1.209 (3.20), 1.224 (1.97), 1.330 (2.14), 1.342 (1.21), 1.37 4 (16.00), 2.477 (0.78), 2.518 (1.19), 2.688 (0.59), 2.847 (3.65), 4.203 (0.84), 4.219 (0.77), 4.259 (0.88), 4.273 (1.08), 4.290 (0.59 ), 4.413 (0.43), 4.430 (0.50), 4.446 (0.51), 4.463 (0.55), 4.481 (0.43), 5.019 (0.52), 5.036 (0.50), 5.754 (3.73), 6.933 (0.63), 7.307 (0.97), 7.325 (2.23), 7.344 (1.63), 7.356 (0.71), 7.396 (1.46), 7.415 (2.36), 7.433 (1.04), 7.603 (0.61), 7.622 (0.61), 7.701 (0.97), 7.719 (1.51), 7.736 (0.70), 7.879 (2.74), 7.898 (2.59). Intermediate 22

L-Alaminyl-N-methyl-L-alanyl-L-aspartate tertiary butyl ester

To a solution of N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-propylaminoyl-N-methyl-L-propylaminoyl-L-aspartic acid tributyl ester (150 mg, 220 µmol) in DMF (2.0 ml) was added DBU (75 µl, 500 µmol; CAS-RN: 6674-22-2) and the reaction was stirred at room temperature for 1 hour. The crude product was used without further purification. Intermediate 23

N-(2-{[(Benzyloxy)carbonyl]amino}ethyl)-N ² ,N ⁶ -bis(tert-butyloxycarbonyl)-L-aminocarboxamide

Prepared as described in WO2016/207089 L108. Intermediate 24

N-(2-aminoethyl)-N ² ,N ⁶ -bis(tert-butyloxycarbonyl)-L-aminoamide

Prepared as described in WO2016/207089 L108. Intermediate 25

N ² ,N ⁶ -bis(tert-butyloxycarbonyl)-N-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethyl}-L-aminocarboxamide

To a solution of N-(2-aminoethyl)-N2,N6-bis(tert-butyloxycarbonyl)-L-carbohydramide (50.0 mg, 129 µmol) in DMF (5.0 ml) were added 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (38.9 mg, 154 µmol) and N,N-diisopropylethylamine (45 µl, 260 µmol; CAS-7087-68-5), and the reaction was stirred at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 51 mg of the product. LC-MS (Method 1): R _t = 0.78 min; MS (ESIpos): m/z = 526 [M+H] ⁺ . Intermediate 26

N-{2-[2-(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethyl}-L-aminocarboxamide

To a solution of N2,N6-bis(tributyloxycarbonyl)-N-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethyl}-L-carboxamide (51.0 mg, 97.0 µmol) in dichloromethane (5.0 ml) was added trifluoroacetic acid (1.0 ml) and the reaction was stirred at room temperature for 2.5 h. The mixture was concentrated under reduced pressure and the residue was diluted in acetonitrile/water and lyophilized to give 33 mg of the product. Intermediate 27

N-(tert-butyloxycarbonyl)-L-hydroxyethyl-L-alanine benzyl ester

N-(tert-butyloxycarbonyl)-L-alanine (500 mg, 2.3 mmol) was dissolved in 10 ml N,N -dimethylformamide, followed by the addition of L-alanine benzyl hydrochloride (496 mg, 2.3 mmol), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylhexafluorophosphate (875 mg, 2.3 mmol) and ethyldiisopropylamine (446 mg, 3.5 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with water. The solid was collected by filtration and the filtrate was washed with water. After drying the filtrate, 550 mg of the product was obtained. Intermediate 28

L-Valamidoyl-L-alanine benzyl ester

N-(tert-Butyloxycarbonyl)-L-carbazyl-L-alanine benzyl ester (550 mg, 1.45 mmol) was dissolved in 20 ml of dichloromethane and then 2 ml of trifluoroacetic acid was added. The reaction mixture was stirred at room temperature for 5 hours. The mixture was evaporated in vacuo and co-distilled 3 times with dichloromethane to give 550 mg (crude) of the product which was used in the next step without further purification. Intermediate 29

1-({N ² -[(Benzyloxy)carbonyl]-N-(17,17-dimethyl-15-oxo-3,6,9,12,16-pentaoxaoctadec-1-yl)-L-glutamidoyl}amino)-3,6,9,12-tetraoxapentadecan-15-oic acid tributyl ester

To a solution of N-[(benzyloxy)carbonyl]-L-glutamine (50.0 mg, 178 µmol) and tributyl 1-amino-3,6,9,12-tetraoxopentadecane-15-ate (126 mg, 391 µmol) in DMF (3.0 ml) were added HATU (162 mg, 427 µmol; CAS-RN: 148893-10-1) and N , N -diisopropylethylamine (120 µl, 710 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 10 min. The mixture was purified by preparative HPLC to give 156 mg of the product. LC-MS (Method 1): R _t = 1.06 min; MS (ESIpos): m/z = 888 [M+H] ⁺ . ¹H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.391 (16.00), 2.390 (0.77), 2.406 (1.62), 2.421 (0.81), 3.162 (0.41), 3.177 (0.45), 3.380 (0.71), 3.395 (0.45), 3.482 (5.36), 3.493 (7.85), 3.562 (1.41), 3.578 (2.15), 3.593 (1.16), 5.012 (0.70), 7.355 (1.17). Intermediate 30

1-({N ² -[(Benzyloxy)carbonyl]-N-(14-carboxy-3,6,9,12-tetraoxotetradec-1-yl)-L-α-glutamidoyl}amino)-3,6,9,12-tetraoxopentadec-15-oic acid

To a solution of tributyl 1-({N2-[(benzyloxy)carbonyl]-N-(17,17-dimethyl-15-oxo-3,6,9,12,16-pentaoxaoctadec-1-yl)-L-α-glutamidoyl}amino)-3,6,9,12-tetraoxapentadecan-15-ate (152 mg, 171 µmol) in dichloromethane (10 ml) was added TFA (530 µl, 6.9 mmol; CAS-RN: 76-05-1) and the reaction was stirred at room temperature for 12 h. The mixture was concentrated to dryness under reduced pressure and co-distilled with dichloromethane (3 times) to give 131 mg of the title product. LC-MS (Method 2): R _t = 1.15 min; MS (ESIpos): m/z = 776 [M+H] ⁺ . ¹H-NMR (400 MHz, DMSO-d6) δ [ppm]: -0.008 (1.74), 0.008 (1.13), 1.073 (0.60), 1.091 (1.22), 1.108 (0.59), 2.418 (1.74), 2.434 (3.65), 2.45 0 (2.06), 2.523 (1.10), 3.164 (0.88), 3.179 (0.98), 3.193 (0.51), 3.357 (0.45), 3.367 (0.89), 3.375 (1.06), 3.382 (1.59), 3.392 (1.23) , 3.397 (1.03), 3.486 (10.96), 3.497 (16.00), 3.577 (2.46), 3.593 (4.43), 3.609 (2.60), 3.681 (1.66), 3.928 (0.51), 3.943 (0.47), 5.013 (1.44), 7.3 56 (2.43), 7.890 (0.48). Intermediate 31

(2S,5S,8S,27S,48S,51S,54S)-2,54-bis(2-amino-2-oxoethyl)-27-{[(benzyloxy)carbonyl]amino}-5,6,8,48,50,51-hexamethyl-4,7,10,26,30,46,49,52-octaoxo-13,16,19,22,34,37,40,43-octaoxa-3,6,9,25,31,47,50,53-octaazapentacontan-1,55-dicarboxylic acid di-tributyl ester

To a solution of 1-({N2-[(benzyloxy)carbonyl]-N-(14-carboxy-3,6,9,12-tetraoxotetradec-1-yl)-L-glutamidoyl}amino)-3,6,9,12-tetraoxopentadec-15-oic acid (38.8 mg, 50.0 µmol) in DMF (1.0 ml) were added HATU (45.7 mg, 120 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (35 µl, 200 µmol; CAS-RN: 7087-68-5), and the mixture was stirred at room temperature for 10 min. A solution of L-propylamino-N-methyl-L-propylamino-L-aspartate (37.9 mg, 110 µmol) in DMF (1 ml, reaction mixture see Intermediate 22) was added and the fraction was stirred for 10 min. Water/0.1% TFA was added and the mixture was purified by preparative HPLC to give 24 mg of product. LC-MS (Method 1): R _t = 0.77 min; MS (ESIneg): m/z = 1426 [MH] ^- . Intermediate 32

(2S,5S,8S,27S,48S,51S,54S)-2,54-bis(2-amino-2-oxoethyl)-27-{[(benzyloxy)carbonyl]amino}-5,6,8,48,50,51-hexamethyl-4,7,10,26,30,46,49,52-octaoxo-13,16,19,22,34,37,40,43-octaoxa-3,6,9,25,31,47,50,53-octaazapentacontan-1,55-dicarboxylic acid

(2S,5S,8S,27S,48S,51S,54S)-2,54-bis(2-amino-2-oxoethyl)-27-{[(benzyloxy)carbonyl]amino}-5,6,8,48,50,51-hexamethyl-4,7,10,26,30,46,49,52-octaoxo-13,16,19,22,34,37,40,43-octaoxa-3,6,9,25,31,47,50,53-octaazapentacontan-1,55-dioic acid di-tributyl ester (24.4 mg, 17.1 µmol) was dissolved in 2,2,2-trifluoroethanol (3 ml). Zinc chloride (14.0 mg, 102 µmol) was added and the reaction was stirred at 50°C. After stirring for 1 hour, zinc chloride (14.0 mg, 102 µmol) was added and the reaction was stirred at 50°C for 1 hour. Zinc chloride (14.0 mg, 102 µmol) was added again and the reaction was stirred at 50°C for another hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (29.9 mg, 102 µmol; CAS-RN: [60-00-4]) was added followed by 2 ml of water/0.1% TFA. The mixture was filtered and the filtrate was purified by preparative HPLC to give 15 mg of product. LC-MS (Method 1): R _t = 0.59 min; MS (ESIneg): m/z = 1314 [MH] ^- . Intermediate 33

N-(2,2-Dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5-azaeicosan-20-yl)-L-hydroxyethyl-L-alanine benzyl ester

2,2-Dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosane-20-oic acid (5 g, 13.7 mmol, CAS-RN: 756525-91-4) was dissolved in N,N-dimethylformamide (42 ml), L-hydroxy-L-alanine benzyl ester (5.3 g, 13.7 mmol), HATU (5.2 g, 13.7 mmol; CAS-RN: 148893-10-1) and ethyldiisopropylamine (2.6 g 20.5 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched with water, and the mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered, and evaporated to dryness. The crude material was purified by column chromatography using dichloromethane/methanol (20:1/v:v) to give 5.0 g of the product. Intermediate 34

N-(15-amino-4,7,10,13-tetraoxopentadec-1-yl)-L-hydroxyethylamino-L-alanine benzyl ester

N-(2,2-Dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxazolidin-5-azaeicosan-20-yl)-L-acetamidoyl-L-alanine benzyl ester (8.00 g, 12.8 mmol) was dissolved in dichloromethane (200 ml), trifluoroacetic acid (20 ml) was added, and the reaction mixture was stirred at room temperature for 5 hours. The mixture was evaporated in vacuo and co-distilled 3 times with dichloromethane to give 8.0 g (crude) of the product, which was used in the next step without further purification. Intermediate 35

(2S,5S,24S,45S,48S)-24-[(tributyloxycarbonyl)amino]-2,48-dimethyl-4,7,23,27,43,46-hexaoxy-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetradecane-1,49-diacid diphenylmethyl ester

N-(tert-Butyloxycarbonyl)-L-glutamine (1.55 g, 6.25 mmol; CAS-RN: 2419-94-5) was dissolved in N,N-dimethylformamide (50 ml), followed by the addition of N-(15-amino-4,7,10,13-tetraoxopentadec-1-yl)-L-glutamidoyl-L-alanine benzyl ester (8.00 g, 12.5 mmol), HATU (4.76 g, 12.5 mmol; CAS-RN: 148893-10-1) and ethyldiisopropylamine (2.4 g, 18.8 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with water, and the mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered, and evaporated to dryness. The crude material was purified by column chromatography using dichloromethane/methanol (20:1/v:v) to give 3.0 g of the product. LC-MS: R _t = 1.49 min; MS (ESIpos): m/z = 402 [M+H] ⁺ intermediate 36

(2S,5S,24S,45S,48S)-24-[(tributyloxycarbonyl)amino]-2,48-dimethyl-4,7,23,27,43,46-hexaoxy-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetradecane-1,49-diacetic acid

(2S,5S,24S,45S,48S)-24-[(tributyloxycarbonyl)amino]-2,48-dimethyl-4,7,23,27,43,46-hexaoxo-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetranonadecan-1,49-dioic acid diphenylmethyl ester (100 mg, 79.2 µmol) was dissolved in methanol (10 ml). Palladium/activated carbon 10% (20 mg) was added. The reaction mixture was stirred at room temperature under an inert atmosphere of hydrogen for 2 h. The crude material was then filtered, washed with methanol and evaporated to dryness to give 70 mg of the product. Intermediate 37

(2S,5S,24S,45S,48S)-24-amino-2,48-dimethyl-4,7,23,27,43,46-hexaoxy-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetradecane-1,49-diacetic acid

(2S,5S,24S,45S,48S)-24-[(tributyloxycarbonyl)amino]-2,48-dimethyl-4,7,23,27,43,46-hexaoxo-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetranonadecan-1,49-dioic acid (70.0 mg, 64.7 µmol) was dissolved in dichloromethane (10 ml), trifluoroacetic acid (0.5 ml) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was evaporated in vacuo and co-distilled with dichloromethane 3 times to give 70 mg of the product, which was used in the next step without further purification. Intermediate 38

(2S,5S,24S,45S,48S)-24-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-2,48-dimethyl-4,7,23,27,43,46-hexaoxo-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetradecane-1,49-diacetic acid

1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (562 mg, 1.82 mmol; CAS-RN: 55750-63-5) was dissolved in N,N-dimethylformamide (50 ml), and (2S,5S,24S,45S,48S)-24-amino-2,48-dimethyl-4,7,23,27,43,46-hexaneoxo-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazatetradecane-1,49-dioic acid (2.00 g, 1.82 mmol) was added. mmol) and ethyldiisopropylamine (354 mg, 2.7 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with water and evaporated to dryness. The crude material was purified by preparative HPLC to give 879 mg of the product. LC-MS: R _t = 1.00 min; MS (ESIpos): m/z = 1175 [M+H] ⁺ intermediate 39

(2S,5S,8S,13S,16S)-8-[(tributyloxycarbonyl)amino]-2,16-dimethyl-4,7,11,14-tetraoxy-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid diphenylmethyl ester

N-(tert-Butyloxycarbonyl)-L-glutamine (173 mg, 701 µmol; CAS-RN: 2419-94-5) was dissolved in N,N-dimethylformamide (10 ml), L-glutamidoyl-L-alanine benzyl ester (550 mg, 1.40 mmol), HATU (533 mg, 1.40 mmol; CAS-RN: [148893-10-1]) and ethyldiisopropylamine (272 mg, 2.10 mmol) were added, and the reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with water. The solid was filtered off and the residue was washed with water. The filtrate was evaporated to dryness to give 400 mg of the product. Intermediate 40

(2S,5S,8S,13S,16S)-8-amino-2,16-dimethyl-4,7,11,14-tetraoxy-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid diphenylmethyl ester

(2S,5S,8S,13S,16S)-8-[(tributyloxycarbonyl)amino]-2,16-dimethyl-4,7,11,14-tetraoxy-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid diphenylmethyl ester (400 mg, 521 µmol) was dissolved in 10 ml dichloromethane and 1.5 ml trifluoroacetic acid was added. The reaction mixture was stirred at room temperature for 5 hours. The mixture was evaporated in vacuo and co-distilled 3 times with dichloromethane to give 400 mg of crude product which was used in the next step without further purification. LC-MS method 5: R _t = 0.97 min; MS (ESIpos): m/z = 668 [M+H] ⁺ intermediate 41

(2S,5S,8S,13S,16S)-8-amino-2,16-dimethyl-4,7,11,14-tetraoxy-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-diacid

(2S,5S,8S,13S,16S)-8-amino-2,16-dimethyl-4,7,11,14-tetraoxy-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid diphenylmethyl ester (200 mg, 256 µmol) was dissolved in 15 ml of methanol. Palladium/activated carbon 10% Pd (50 mg) was added and the reaction mixture was stirred at room temperature for 2 h under an inert atmosphere of hydrogen. The mixture was then filtered and the solid was washed with methanol. The filtrate was evaporated to dryness under reduced pressure to give 100 mg of the product. Intermediate 42

(2S,5S,8S,13S,16S)-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-2,16-dimethyl-4,7,11,14-tetraoxo-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid

1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (1.14 g, 3.69 mmol; CAS-RN: 55750-63-5) was dissolved in 50 ml of N,N-dimethylformamide, (2S,5S,8S,13S,16S)-8-amino-2,16-dimethyl-4,7,11,14-tetraoxo-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid (1.50 g, 3.08 mmol) and ethyldiisopropylamine (398 mg, 3.1 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours. The reaction was quenched with water and evaporated to dryness. The crude material was purified twice by preparative HPLC to give 260 mg of product. LC-MS: R _t = 1.02 min; MS (ESIpos): m/z = 682 [M+H] ⁺ . Intermediate 43

N ² ,N ⁶ -bis[(benzyloxy)carbonyl]-N-{2-[(tert-butyloxycarbonyl)amino]ethyl}-L-aminocarboxamide

To a solution of N2,N6-bis[(benzyloxy)carbonyl]-L-lysine (30.0 mg, 72.4 µmol) and (2-aminoethyl)carbamic acid tert-butyl ester (13 µl, 80 µmol) in DMF (5.0 ml) were added HATU (33.0 mg, 86.9 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (38 µl, 220 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure, and the residue was dissolved in a mixture of acetonitrile/water/dioxane and purified by preparative HPLC to give 35 mg of the product. LC-MS (method 1): R _t = 1.02 min; MS (ESIpos): m/z = 557 [M+H] ⁺ . Intermediate 44

N-{2-[(tert-butyloxycarbonyl)amino]ethyl}-L-aminocarboxamide

To a solution of N2,N6-bis[(benzyloxy)carbonyl]-N-{2-[(tert-butyloxycarbonyl)amino]ethyl}-L-diaminobenzamide (35.0 mg, 62.9 µmol) in ethanol (10 ml) was added 10% palladium/activated carbon (5 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 21 mg of the title product. LC-MS (Method 1): R _t = 0.16 min; MS (ESIpos): m/z = 289 [M+H] ⁺ . KSPi intermediate intermediate 45

(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine

The title compound was prepared as described in WO2006/002326. Intermediate 46

N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

The title compound was prepared as described in WO2015/096982. Intermediate 47

(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-[(tert-butyloxycarbonyl)amino]butanoic acid

The title compound was prepared as described in WO2016/207089. Intermediate 48

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester

The title compound was prepared as described in WO2018/114798. Intermediate 49

N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}amino)ethyl]-N ² -[(benzyloxy)carbonyl]-D-α-glutamylamide benzyl ester

The title compound was prepared as described in WO2016/207089. Intermediate 50

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tributyl ester

The title compound was prepared as described in WO2018/114798. Intermediate 51

(3R,10S,13S,16S,35S,56S,59S,62S,69R)-10,62-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-35-{[6-(2,5-dihydro-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}- 13,59-Dimethyl-5,9,12,15,18,34,38,54,57,60,63,67-dodecyloxy-16,56-di(propan-2-yl)-21,24,27,30,42,45,48,51-octaoxana-4,8,11,14,17,33,39,55,58,61,64,68-dodecanoic acid tetratributyl ester

To (2S,5S,24S,45S,48S)-24-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-2,48-dimethyl-4,7,23,27,43,46-hexaoxo-5,45-di(propan-2-yl)-10,13,16,19,31,34,37,40-octaoxa-3,6,22,28,44,47-hexaazanonadecadecane-1,49-dioic acid (20.0 mg, 17.0 To a solution of 2-[(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (28.1 mg, 34.0 µmol) in DMF (2.0 ml) were added HATU (15.5 mg, 40.8 µmol; CAS-RN:148893-10-1) and N,N-diisopropylethylamine (12 µl, 68 µmol; CAS-RN:7087-68-5), and the reaction was stirred at room temperature for 10 min. Ethyl acetate was added to the mixture, and the mixture was washed 3 times with brine (20 ml). The organic phase was dried over magnesium sulfate, filtered, and evaporated to dryness. The crude material was purified by column chromatography to give 32 mg of the product. LC-MS (Method 4): R _t = 7.71 min; MS (ESIpos): m/z = 1418 [M+2Na] ²⁺ . Intermediate 52

(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid

The title compound was prepared as described in WO2015/096982. Intermediate 53

2-(Trimethylsilyl)ethyl [(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]carbamate

To a solution of (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid (400 mg, 608 µmol) in DMF (67 ml) were added N-methyl-β-propylaminoamide (68.3 mg, 669 µmol), HATU (277 mg, 730 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (320 µl, 1.8 mmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 368 mg of the product. LC-MS (Method 1): R _t = 1.30 min; MS (ESIpos): m/z = 742 [M+H] ⁺ . Intermediate 54

(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-N-[3-(methylamino)-3-oxopropyl]butyramide

2-(Trimethylsilyl)ethyl [(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]carbamate (368 mg, 496 µmol) was dissolved in 2,2,2-trifluoroethanol (8 ml). Zinc chloride (406 mg, 2.98 mmol) was added and the reaction was stirred at 50° C. for 2 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (870 mg, 2.98 mmol; CAS-RN: 60-00-4) was added, followed by 2 ml of water/0.1% TFA, and the mixture was stirred for several minutes. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 306 mg of product. LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 598 [M+H] ⁺ . Intermediate 55

(2R)-2-{[(5S,8S,11S,14S)-11-(2-amino-2-oxoethyl)-14-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,7,8-trimethyl-3,6,9,12,15,19-hexaoxo-1-phenyl-2-oxo-4,7,10,13,16-pentazanonadecan-19-yl]amino}diphenylmethyl glutarate

The title compound was prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 56

L-propylaminoyl-N-methyl-L-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

The title compound was prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 57

N-(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohex-2-yl]amino}-5-oxopentanyl)-L-propylamino-N-methyl-L-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

To a solution of N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylaminoyl-N-methyl-L-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (36.0 mg, 30.1 µmol) in DMF (8.5 ml) was added N,N-diisopropylethylamine (10 µl, 60 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 20 hours. The reaction was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 31 mg of the product. LC-MS (Method 4): R _t = 1.12 min; MS (ESIpos): m/z = 1416 [M+H] ⁺ . Intermediate 58

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-propylaminoyl-N-methyl-L-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

The title compound was prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 59

N-(5-{[(2S)-6-amino-1-tributyloxy-1-oxohexyl-2-yl]amino}-5-oxopentanyl)-L-propylamino-N-methyl-L-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

N-(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanyl)-L-propylamino-N-methyl-L-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide (31.0 mg, 21.9 µmol) in methanol/DCM (25 10% palladium/activated carbon (8.2 mg) was added to a solution of 4% paraffin (2% paraffin) in 1% paraffin (4% paraffin) (ml, 1:1). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and freeze-dried to give 28 mg of the title product. LC-MS (Method 1): R _t = 0.84 min; MS (ESIpos): m/z = 1282 [M+H] ⁺ . Intermediate 60

(8S,11S,14S,17S,24R)-14-(2-amino-2-oxoethyl)-17-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-24-{[(benzyloxy)carbonyl]amino}-2,2,8,11-tetramethyl-6,9,12,15,18,23-hexaoxo-5-oxo-7,10,13,16,19,22-hexaaza-2-silascosa-27-oic acid benzyl ester

To a solution of N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}amino)ethyl]-N2-[(benzyloxy)carbonyl]-D-α-glutamidobenzyl ester (213 mg, 208 µmol) and N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-propylamino-L-propylamino-L-aspartic acid (122 mg, 291 µmol) in DMF (15 ml) was added HATU (158 mg, 416 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (110 µl, 620 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 5 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 202 mg of the product. LC-MS (Method 1): R _t = 1.37 min; MS (ESIpos): m/z = 1309 [M+H] ⁺ . Intermediate 61

N-{[2-(Trimethylsilyl)ethoxy]carbonyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-({2-[(D-α-glutamidoyl)amino]ethyl}amino)-1-oxobutyl-2-yl]-L-aspartamide

To a solution of (8S,11S,14S,17S,24R)-14-(2-amino-2-oxoethyl)-17-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-24-{[(benzyloxy)carbonyl]amino}-2,2,8,11-tetramethyl-6,9,12,15,18,23-hexaoxo-5-oxa-7,10,13,16,19,22-hexaaza-2-silascosa-27-oate (202 mg, 154 µmol) in methanol (20 ml) was added 10% palladium/activated carbon (20 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 127 mg of the title product. LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 1085 [M+H] ⁺ . Intermediate 62

N-{[2-(Trimethylsilyl)ethoxy]carbonyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutyl-2-yl]-L-aspartamide

N-{[2-(Trimethylsilyl)ethoxy]carbonyl}-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-({2-[(D-α-glutamidoyl)amino]ethyl}amino)-1-oxobutyl-2-yl]-L-aspartamide (127 mg, 117 µmol) and 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (41.3 mg, 164 µmol) in DMF (14 N,N-diisopropylethylamine (61 µl, 350 µmol; CAS-RN: 7087-68-5) was added to a solution of 4% paraformaldehyde (20% paraformaldehyde) in 1% paraformaldehyde (10% paraformaldehyde) and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 107 mg of the product. LC-MS (Method 1): R _t = 1.13 min; MS (ESIpos): m/z = 1223 [M+H] ⁺ . Intermediate 63

L-propylaminoyl-L-propylaminoyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutyl-2-yl]-L-aspartamide

N-{[2-(Trimethylsilyl)ethoxy]carbonyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutan-2-yl]-L-aspartamide (107 mg, 87.5 µmol) was dissolved in 2,2,2-trifluoroethanol (10 ml). Zinc chloride (71.6 mg, 525 µmol; CAS-RN: 7646-85-7) was added and the reaction was stirred at 50 °C for 5 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (153 mg, 525 µmol; CAS-RN: [60-00-4]) was added followed by acetonitrile/water. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 77 mg of the title product. LC-MS (Method 1): R _t = 0.81 min; MS (ESIpos): m/z = 1078 [M+H] ⁺ . Intermediate 64

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide

L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutyl-2-yl]-L-aspartamide) (180 mg, 151 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (148 mg, 453 µmol) in DMF (15 ml) was added N,N-diisopropylethylamine (79 µl, 450 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 159 mg of the product. LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 1290 [M+H] ⁺ . Intermediate 65

(3R,10S,13S,16S,19S,27S,39S,42S,45S,48S,55R)-13,45-bis(2-amino-2-oxoethyl)-10,48-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-27-(tert-butyloxycarbonyl)-55-[2 [(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-16,17,19,39,42-pentamethyl-5,9,12,15,18,21,25,33,37,40,43,46,49,54-tetradecanoyl-4,8,11,14,17,20,26,32,38,41,44,47,50,53-tetradecanoheptadecane-1,3,57-tricarboxylic acid]

N-(5-{[(2S)-6-amino-1-tributyloxy-1-oxohex-2-yl]amino}-5-oxopentanyl)-L-propylamino-N-methyl-L-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide (24.9 mg, 19.4 µmol) in DMF (8.3 To a solution of 14.5 ml (25.0 mg, 19.4 mL) was added N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide (25.0 mg, 19.4 mL) and 4.5 ml (25.0 mg, 19.4 mL) and 4.5 ml of N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-propylamino-N 1 -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide (25.0 mg, 19.4 mL) and 4.5 ml of N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-N 1 -[(2S)-4-[{(1 µmol) and N,N -diisopropylethylamine (10 µl, 58 µmol; CAS-RN7087-68-5) were added and the reaction was stirred at room temperature for 20 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 32 mg of the product. LC-MS (Method 1): R _t = 1.11 min; MS (ESIpos): m/z = 1228 [M+2H] ²⁺ .Intermediate 66

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(tert-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylamino-D-glutamine diphenylmethyl ester

Compounds were prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 67

N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine diphenylmethyl ester

Compounds were prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 68

N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine

Compounds were prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 69

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-[(N ² -{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-asparagine)amino]butyryl}-β-propylamino-D-glutamine

Compounds were prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 70

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanyl)-L-asparagine]amino}butyryl]-β-propylamino-D-glutamine

To a solution of N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-[(N2-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-asparagine)amino]butyryl}-β-propylaminoyl-D-glutamine (38.0 mg, 36.6 µmol) in DMF (10 ml) were added N6-[(benzyloxy)carbonyl]-L-lysine tributyl ester (14.8 mg, 43.9 µmol) and N,N -diisopropylethylamine (13 µl, 73 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 20 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 32 mg of the product. LC-MS (Method 1): R _t = 1.12 min; MS (ESIpos): m/z = 1260 [M+H] ⁺ . Intermediate 71

N-{(2S)-2-{[N ² -(5-{[(2S)-6-amino-1-tributyloxy-1-oxohexyl-2-yl]amino}-5-oxohexylpentanoyl)-L-asparagine]amino}-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine

To a solution of N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[ ^N2- (5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanyl)-L-asparagine]amino}butyryl]-β-propylaminoyl-D-glutamine (32.0 mg, 25.4 µmol) in methanol/DCM (25 ml, 1:1) was added 10% palladium/activated carbon (10 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 29 mg of the title product. LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 1126 [M+H] ⁺ . Intermediate 72

(3R,10S,13S,21S,33S,36S,39S,42S,49R)-13,39-bis(2-amino-2-oxoethyl)-10,42-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-21-(tert-butyloxycarbonyl) )-49-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-33,36-dimethyl-5,9,12,15,19,27,31,34,37,40,43,48-dodecanoyl-4,8,11,14,20,26,32,35,38,41,44,47-dodecahydro-1,3,51-tricarboxylic acid

N-{(2S)-2-{[N ² -(5-{[(2S)-6-amino-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanyl)-L-asparagine]amino}-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine (18.0 mg, 16.0 µmol) in DMF (7.0 To a solution of 147 mL) was added N-{5-[(2,5-dioxo-1H-pyrrolidin-1-yl)oxy]-5-oxo-pentanoyl}-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-oxo-2-butyl]-L-aspartamide (20.6 mg, 16.0 mL). µmol) and N,N -diisopropylethylamine (8.4 µl, 48 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 20 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 14 mg of the product. LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 1151 [M+2H] ²⁺ . Intermediate 73

N-{3-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyloxy}ethoxy)ethoxy]propionyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide

L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutyl-2-yl]-L-aspartamide (80.0 mg, 61.2 To a solution of 1,1'-{oxybis[(ethane-2,1-diyl)oxy(1-oxopropane-3,1-diyl)oxy]}bis(pyrrolidine-2,5-dione) (81.7 mg, 184 µmol) in DMF (2.0 ml) was added N,N-diisopropylethylamine (43 µl, 240 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1 hour. A mixture of water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 56 mg of the product. LC-MS (Method 5): R _t = 4.16 min; MS (ESIpos): m/z = 1407 [M+H] ⁺ . Intermediate 74

L-propylaminoyl-L-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

The title compound was prepared as described in Bioconjugate Chem. 2020, 31, 1893−1898. Intermediate 75

N-(3-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyloxy}propionyl)-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide

L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutyl-2-yl]-L-aspartamide (100 mg, 76.6 To a solution of 1,1'-{oxybis[(1-oxopropane-3,1-diyl)oxy]}bis(pyrrolidine-2,5-dione) (81.8 mg, 230 µmol) in DMF (3.0 ml) was added N,N -diisopropylethylamine (53 µl, 310 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1 hour. Water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 63 mg of the product. LC-MS (Method 1): R _t = 0.94 min; MS (ESIneg): m/z = 1317 [MH] ^- . Intermediate 76

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(tert-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylamino-D-glutamine di-tert-butyl ester

To a solution of N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (100 mg, 121 µmol) and N2-(tert-butyloxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidin-1-yl ester (43.9 mg, 133 µmol; CAS-RN:42002-18-6) in DMF (3.0 ml) was added N,N-diisopropylethylamine (63 µl, 360 µmol; CAS-RN: [7087-68-5]), and the reaction was stirred at room temperature for 3 days. Water/0.1% TFA was added, and the mixture was purified by preparative HPLC to give 89 mg of product. LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 1040 [M+H] ⁺ . Intermediate 77

N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N2-(t-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylaminoyl-D-glutamine di-t-butyl ester (89.2 mg, 85.8 µmol) was dissolved in 2,2,2-trifluoroethanol (3 ml). Zinc chloride (70.1 mg, 515 µmol) was added and the reaction was stirred at 50°C. After stirring for 1 hour, zinc chloride (70.1 mg, 515 µmol) was added and the reaction was stirred at 50°C. After one hour, more zinc chloride (70.1 mg, 515 µmol) was added, and the reaction was stirred at 50 °C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (150 mg, 515 µmol; CAS-RN: [60-00-4]) was added, and the mixture was stirred for several minutes. The reaction was filtered and the solid was washed with acetonitrile. The filtrate was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 37 mg of product. LC-MS (Method 1): R _t = 0.78 min; MS (ESIpos): m/z = 828 [M+H] ⁺ . Intermediate 78

(8S,11S,14S)-11-(2-amino-2-oxoethyl)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-14-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-3,7,10,13-tetraoxo-2,6,9,12-tetraazahexadecane-16-oic acid benzyl ester

To a solution of (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-N-[3-(methylamino)-3-oxopropyl]butyramide (100 mg, 141 µmol) in DMF (15 ml) were added (5S,8S,11S)-11-(2-amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecane-12-oic acid (98.6 mg, 155 µmol), HATU (64.1 mg, 169 µmol; CAS-RN: 148893-10-1) and N,N -diisopropylethylamine (73 µl, 420 µmol; CAS-RN: [7087-68-5]) were added and the reaction was stirred at room temperature for 60 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 112 mg of the product. LC-MS (Method 1): R _t = 1.16 min; MS (ESIpos): m/z = 1122 [M+H] ⁺ . Intermediate 79

L-propylaminoyl-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]-L-asparaginyl

To a solution of (8S,11S,14S)-11-(2-amino-2-oxoethyl)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-14-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-3,7,10,13-tetraoxo-2,6,9,12-tetraazahexadecane-16-oic acid benzyl ester (112 mg, 99.8 µmol) in methanol/DCM (20 ml, 1:1) was added 10% palladium/activated carbon (20 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 80 mg of the title product. LC-MS (Method 1): R _t = 0.80 min; MS (ESIpos): m/z = 898 [M+H] ⁺ . Intermediate 80

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-propylamino-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]-L-asparaginyl

To a solution of L-propylamino-L-α-asparaginyl-N1-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutan-2-yl]-L-asparagine (80.0 mg, 89.1 µmol) in DMF (10 ml) were added 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (72.7 mg, 223 µmol) and N,N -diisopropylethylamine (47 µl, 270 µmol; CAS-RN: 7087-68-5) and stirred the reaction at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 57 mg of the product. LC-MS (Method 1): R _t = 0.91 min; MS (ESIpos): m/z = 1109 [M+H] ⁺ . Intermediate 81

N-(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohex-2-yl]amino}-5-oxopentanyl)-L-propylamino-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]-L-asparaginyl

To a solution of N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutan-2-yl]-L-asparagine (57.0 mg, 51.4 µmol) in DMF (5.0 ml) was added N ⁶ -[(benzyloxy)carbonyl]-L-lysine tributyl ester (20.7 mg, 61.7 µmol) and N,N -diisopropylethylamine (18 µl, 100 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 3 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 58 mg of the product. LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 1331 [M+H] ⁺ . Intermediate 82

N-(5-{[(2S)-6-amino-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanoyl)-L-propylamino-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutan-2-yl]-L-asparaginyl

N-(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanyl)-L-propylamino-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutan-2-yl]-L-asparaginyl (58.0 mg, 43.6 µmol) in methanol/DCM (10 10% palladium/activated carbon (9.5 mg) was added to a solution of 4% parathion (2% parathion, 1:1) in 4% parathion (2:1). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and freeze-dried to give 52 mg of the title product. LC-MS (Method 1): R _t = 0.86 min; MS (ESIneg): m/z = 1194 [MH] ^- . Intermediate 83

(3S,6S,14S,26S,29S,32S,35S,42R)-3-{[(2S)-4-amino-1-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]amino}-1,4-dioxobutyl-2-yl]aminomethyl}-32-(2-amino-2-oxoethyl)-35-{2- [{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-14-(t-butyloxycarbonyl)-42-[2-(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-6,26,29-trimethyl-5,8,12,20,24,27,30,33,36,41-decaazapentatetradecan-1,45-diol

N-(5-{[(2S)-6-amino-1-tributyloxy-1-oxohex-2-yl]amino}-5-oxopentanyl)-L-propylamino-L-α-asparaginyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobut-2-yl]-L-asparaginyl (26.4 mg, 22.1 µmol) in DMF (5.0 To a solution of 14.7 ml) was added N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide (19.0 mg, 14.7 ml). µmol) and N,N-diisopropylethylamine (5.1 µl, 29 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 3 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 15 mg of the product. LC-MS (Method 1): R _t = 1.12 min; MS (ESIneg): m/z = 1183 [M-2H] ^2- . Intermediate 84

N-[(Benzyloxy)carbonyl]-L-propylaminoyl-L-propylaminoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine

To a solution of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (125 mg, 214 µmol) in DMF (17 ml) were added N-[(benzyloxy)carbonyl]-L-propylamidoyl-L-propylamidoyl-L-aspartic acid (96.2 mg, 236 µmol), HATU (244 mg, 643 µmol; CAS-RN: 148893-10-1) and N,N -diisopropylethylamine (150 µl, 860 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 122 mg of product. LC-MS (Method 1): R _t = 1.17 min; MS (ESIpos): m/z = 860 [M+H] ⁺ . Intermediate 85

L-propylaminoyl-L-propylaminoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine

To a solution of N-[(benzyloxy)carbonyl]-L-propylaminoyl-L-propylaminoyl-N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine (122 mg, 142 µmol) in methanol/DCM (30 ml, 1:1) was added 10% palladium/activated carbon (10 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 120 mg of the title product. LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 726 [M+H] ⁺ . Intermediate 86

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide

To a solution of L-propylaminoyl-L-propylaminoyl-N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (35.0 mg, 48.2 µmol) in DMF (4.0 ml) were added 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (23.6 mg, 72.3 µmol) and N,N-diisopropylethylamine (17 µl, 96 µmol; CAS-RN:7087-68-5), and the reaction was stirred at room temperature for 2 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 30 mg of the product. LC-MS (Method 1): R _t = 1.02 min; MS (ESIpos): m/z = 937 [M+H] ⁺ . Intermediate 87

N-(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohexyl-2-yl]amino}-5-oxopentanyl)-L-propylamino-L-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine

To a solution of N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (18.5 mg, 17.6 µmol) in DMF (2.0 ml) were added N ⁶ -[(benzyloxy)carbonyl]-L-lysine tributyl ester-hydrochloride (6.56 mg, 17.6 µmol) and N,N -diisopropylethylamine (6.1 µl, 35 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 11 mg of the product. LC-MS (Method 1): R _t = 1.20 min; MS (ESIpos): m/z = 1158 [M+H] ⁺ . Intermediate 88

N-(5-{[(2S)-6-amino-1-tributyloxy-1-oxohexyl-2-yl]amino}-5-oxohexylpentanoyl)-L-propylamino-L-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine

To a solution of N-(5-{[(2S)-6-{[(benzyloxy)carbonyl]amino}-1-tributyloxy-1-oxohexan-2-yl]amino}-5-oxopentanyl)-L-propylamino-L-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (11.2 mg, 9.67 µmol) in methanol/DCM (10 ml, 1:1) was added 10% palladium/activated carbon (4 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 10 mg of the title product. LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 1124 [M+H] ⁺ . Intermediate 89

N-(5-{[(1S)-5-amino-1-carboxypentyl]amino}-5-oxopentanyl)-L-propylamino-L-propylamino-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide trifluoroacetate

N-(5-{[(2S)-6-amino-1-tributyloxy-1-oxohexyl-2-yl]amino}-5-oxopentanyl)-L-propylamino-L-propylamino-N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (9.90 mg, 9.67 µmol) was dissolved in 2,2,2-trifluoroethanol (4 ml). Zinc chloride (7.90 mg, 58.0 µmol) was added and the reaction was stirred at 50°C for 3 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (16.9 mg, 58.0 µmol; CAS-RN: 60-00-4) was added, followed by 2 ml of water/0.1% TFA, and the mixture was purified by preparative HPLC to give 12 mg of product. LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 968 [M+H] ⁺ . Intermediate 90

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(tert-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylamino-D-glutamine diphenylmethyl ester

To a solution of trifluoroacetic acid-N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (370 mg, 367 µmol) and N2-(tert-butyloxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidin-1-yl ester (133 mg, 404 µmol; CAS-RN:42002-18-6) in DMF (6.8 ml) was added N,N-diisopropylethylamine (190 µl, 1.1 mmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1.5 hours. Water/0.1% TFA was added and the mixture was purified by preparative HPLC to give 310 mg of product. LC-MS (Method 2): R _t = 2.54 min; MS (ESIpos): m/z = 1108 [M+H] ⁺ . Intermediate 91

N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine diphenylmethyl ester

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N2-(t-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylaminoyl-D-glutamine diphenylmethyl ester (1.02 g, 917 µmol) was dissolved in 2,2,2-trifluoroethanol (20 ml). Zinc chloride (750 mg, 5.50 mmol) was added and the reaction was stirred at 50°C for 30 min. 10 ml of water/acetonitrile (1:1), TFA (100 µl) and ethylenediamine-N,N,N',N'-tetraacetic acid (1.61 g, 5.50 mmol; CAS-RN: 60-00-4) were added and the mixture was stirred for several minutes. The mixture was filtered, the filtrate was concentrated under reduced pressure and purified by preparative HPLC to give 745 mg of the product. LC-MS (Method 1): R _t = 1.05 min; MS (ESIpos): m/z = 1008 [M+H] ⁺ . Intermediate 92

(2R)-2-{[(6S,9S,12S,15S)-12-(2-amino-2-oxoethyl)-15-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-2,2,6,8,9-pentamethyl-4,7,10,13,16,20-hexaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-20-yl]amino}diphenylmethyl glutarate

To a solution of N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (212 mg, 189 µmol) and N-(tert-butyloxycarbonyl)-L-propylaminoyl-N-methyl-L-alanine (62.2 mg, 227 µmol) in DMF (5.0 ml) were added HATU (86.2 mg, 227 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (99 µl, 570 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 10 min. The mixture was filtered and the filtrate was purified by preparative HPLC to give 166 mg of the product. LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 1265 [M+H] ⁺ . Intermediate 93

(2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12,13-dimethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}diphenylmethyl glutarate

(2R)-2-{[(6S,9S,12S,15S)-12-(2-amino-2-oxoethyl)-15-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-2,2,6,8,9-pentamethyl-4,7,10,13,16,20-hexaneoxo-3-oxa-5,8,11,14,17-pentaazaicostan-20-yl]amino}pentanedioic acid diphenylmethyl ester (213 mg, 168 µmol) was dissolved in 2,2,2-trifluoroethanol (7 ml). Zinc chloride (138 mg, 1.01 mmol) was added and the reaction was stirred at 50 °C for 1.5 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (295 mg, 1.01 mmol; CAS-RN: 60-00-4) was added and the mixture was stirred for a few minutes before water/0.1% TFA was added. The mixture was filtered and concentrated under reduced pressure and the residue was purified by preparative HPLC to give 189 mg of product. LC-MS (Method 3): R _t = 3.56 min; MS (ESIpos): m/z = 1164 [M+H] ⁺ . Intermediate 94

(2R)-2-{[(10S,13S,16S,19S,22S)-19-(2-amino-2-oxoethyl)-22-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-10-{[(benzyloxy)carbonyl]amino}-2,2,13,15,16-pentamethyl-4,11,14,17,20,23,27-heptanoyloxy-3-oxa-5,12,15,18,21,24-hexaazaheptacosan-27-yl]amino}diphenylmethyl glutarate

To (2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12,13-dimethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}pentanedioic acid diphenylmethyl ester (30.0 mg, 23.5 µmol) and N ² -[(benzyloxy)carbonyl]-N ⁶ -(t-butyloxycarbonyl)-L-lysine (10.7 mg, 28.2 µmol) in DMF was added distilled water and the mixture was stirred for 2 h. HATU (10.7 mg, 28.2 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (12 µl, 70 µmol; CAS-RN: 7087-68-5) were added to a solution of 4-nitropropene (6.0 ml) and the reaction was stirred at room temperature for 60 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 32 mg of the product. LC-MS (Method 1): R _t = 1.39 min; MS (ESIpos): m/z = 1525 [M+H] ⁺ . Intermediate 95

N ⁶ -(tert-butyloxycarbonyl)-L-aminoyl-L-propylaminoyl-N-methyl-L-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

To (2R)-2-{[(10S,13S,16S,19S,22S)-19-(2-amino-2-oxoethyl)-22-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-10-{[(benzyloxy)carbonyl]amino}-2,2,13,15,16-pentamethyl-4,11,14,17,20,23,27-heptadoxy-3-oxa-5,12,15,18,21,24-hexaazaheptacosane-27-yl]amino}diphenylmethyl glutarate (49.0 mg, 32.1 To a solution of 20 μmol (40 μg) in methanol (10 ml) was added 10% palladium/activated carbon (5 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 3 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 39 mg of the title product. LC-MS (Method 1): R _t = 0.87 min; MS (ESIpos): m/z = 1212 [M+H] ⁺ . Intermediate 96

[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]carbamic acid benzyl ester

To a solution of N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1.00 g, 1.71 mmol) in N,N-diisopropylethylamine (600 µl, 3.4 mmol; CAS-RN: 7087-68-5) was added N2-[(benzyloxy)carbonyl]-L-aspartic acid 4-nitrophenyl ester (1.19 g, 3.08 mmol) and DMF (50 ml), and the reaction was stirred at room temperature for 20 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 975 mg of the product. LC-MS (method 1): R _t = 1.21 min; MS (ESIpos): m/z = 718 [M+H] ⁺ . Intermediate 97

N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine

To a solution of benzyl [(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutan-2-yl]carbamate (975 mg, 1.36 mmol) in methanol/DCM (97 ml, 1:1) was added 10% palladium/activated carbon (95 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 763 mg of the title product. LC-MS (method 1): R _t = 0.82 min; MS (ESIpos): m/z = 584 [M+H] ⁺ . Intermediate 98

(3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(benzyloxy)carbonyl]amino}-4-oxobutyl acid tributyl ester

To a solution of N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (763 mg, 1.31 mmol) in DMF (75 ml) were added (2S)-2-{[(benzyloxy)carbonyl]amino}-4-tributyloxy-4-oxobutanoic acid (549 mg, 1.70 mmol), HATU (746 mg, 1.96 mmol; CAS-RN:148893-10-1) and N,N-diisopropylethylamine (460 µl, 2.6 mmol; CAS-RN:[7087-68-5]), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 914 mg of product. LC-MS (Method 1): R _t = 1.28 min; MS (ESIpos): m/z = 889 [M+H] ⁺ . Intermediate 99

(3S)-3-amino-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-4-oxobutyl acid tributyl ester

To a solution of (3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(benzyloxy)carbonyl]amino}-4-oxobutanoic acid tributyl ester (914 mg, 1.03 mmol) in methanol/DCM (75 ml, 1:1) was added 10% palladium/activated carbon (50 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 814 mg of the title product. LC-MS (Method 1): R _t = 0.89 min; MS (ESIpos): m/z = 755 [M+H] ⁺ . Intermediate 100

(3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-4-oxobutyl acid tributyl ester

To a solution of (3S)-3-amino-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-4-oxobutyric acid tributyl ester (841 mg, 1.11 mmol) in DMF (64 ml) were added N-[(benzyloxy)carbonyl]-L-alanine (323 mg, 1.45 mmol), HATU (635 mg, 1.67 mmol; CAS-RN:148893-10-1) and N,N -diisopropylethylamine (390 µl, 2.2 mmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 964 mg of the product. LC-MS (Method 1): R _t = 1.25 min; MS (ESIpos): m/z = 960 [M+H] ⁺ . Intermediate 101

(3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-aminopropionyl]amino}-4-oxobutyl acid tributyl ester

To a solution of (3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-4-oxobutanoic acid tributyl ester (964 mg, 1.00 mmol) in methanol/DCM (73 ml, 1:1) was added 10% palladium/activated carbon (49 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 788 mg of the title product. LC-MS (Method 1): R _t = 0.88 min; MS (ESIpos): m/z = 826 [M+H] ⁺ . Intermediate 102

L-propylaminoyl-L-α-asparaginyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparaginyl-trifluoroacetic acid (1/1)

(3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-aminopropionyl]amino}-4-oxobutyric acid tributyl ester (109 mg, 132 µmol) was dissolved in 2,2,2-trifluoroethanol (11 ml). Zinc chloride (108 mg, 792 µmol) was added and the reaction was stirred at 50°C for 4 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (231 mg, 792 µmol; CAS-RN: [60-00-4]) was added, followed by 2 ml of water/0.1% TFA. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 72 mg of product. LC-MS (Method 1): R _t = 0.83 min; MS (ESIpos): m/z = 770 [M+H] ⁺ intermediate 103

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-α-asparaginyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparaginyl

To a solution of L-propylaminoyl-L-α-asparaginyl-N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine (34.0 mg, 38.5 µmol) in DMF (6.0 ml) were added 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (50.2 mg, 154 µmol) and N,N-diisopropylethylamine (20 µl, 120 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 13 mg of product. LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m/z = 981 [M+H] ⁺ intermediate 104

(2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-22-(2-amino-2-oxoethyl)-25-{2-[{(1R) -1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13-{4-[(tert-butyloxycarbonyl)amino]butyl}-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonazolyl-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-α-asparaginyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparaginyl (20.5 mg, 20.9 µmol) and N ⁶ -(tert-butyloxycarbonyl)-L-hydroxyamino-L-propylamino-N-methyl-L-propylamino-N ¹ To a solution of -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (32.5 mg, 23.2 µmol) in DMF (5.0 ml) was added N,N -diisopropylethylamine (12 µl, 70 µmol; CAS-RN:7087-68-5) and the reaction was stirred at room temperature for 4 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 13 mg of product. LC-MS (Method 1): R _t = 1.21 min; MS (ESIneg): m/z = 1037 [M-2H] ^2- Intermediate 105

(2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-13-(4-aminobutyl)-2,2-(2-amino-2-oxobutyl)- 2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonazolyl-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid

(2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl}-22-(2-amino-2-oxoethyl)-25-{2-[{(1R)-1 1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13-{4-[(t-butyloxycarbonyl)amino]butyl}-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonaoxo-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid (12.5 mg, 6.01 µmol) was dissolved in 2,2,2-trifluoroethanol (5 ml). Zinc chloride (4.9 mg, 36.1 µmol) was added and the reaction was stirred at 50°C for 5 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (10.5 mg, 36.1 µmol; CAS-RN: [60-00-4]) was added, followed by water/0.1% TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 11 mg of product. LC -MS (Method 1): R _t = 1.03 min; MS (ESIneg): m/z = 1176 [MH] ^{-Intermediate} 106

(2R)-2-{[(5S,8R,11S,14S)-11-(2-amino-2-oxoethyl)-14-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,8-dimethyl-3,6,9,12,15,19-hexaoxo-1-phenyl-2-oxo-4,7,10,13,16-pentazanonadecan-19-yl]amino}diphenylmethyl glutarate

To a solution of N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (120 mg, 119 µmol) in DMF (15 ml) were added N-[(benzyloxy)carbonyl]-L-propylaminoyl-D-propylaminoyl-L-aspartic acid (74.6 mg, 143 µmol), HATU (67.9 mg, 179 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (62 µl, 360 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 62 mg of the product. LC-MS (Method 1): R _t = 1.33 min; MS (ESIpos): m/z = 1284 [M+H] ^2-+ Intermediate 107

L-propylaminoyl-D-propylaminoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

To a solution of (2R)-2-{[(5S,8R,11S,14S)-11-(2-amino-2-oxoethyl)-14-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,8-dimethyl-3,6,9,12,15,19-hexaoxo-1-phenyl-2-oxazolidin-4,7,10,13,16-pentazanonadecan-19-yl]amino}pentanedioic acid diphenylmethyl ester (78.4 mg, 61.0 µmol) in methanol/DCM (25 ml, 1:1) was added 10% palladium/activated carbon (15 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 64 mg of the title product.

LC-MS (Method 1): R _t = 0.78 min; MS (ESIneg): m/z = 968 [MH] ^{-Intermediate} 108

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-propylamino-D-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

L-propylamino-D-propylamino-N1-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (69.0 mg, 71.1 µmol) and 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (58.0 mg, 178 µmol) in DMF (6.9 N,N-diisopropylethylamine (37 µl, 210 µmol; CAS-RN: 7087-68-5) was added to a solution of 4% paraformaldehyde (20% paraformaldehyde) in 1% paraformaldehyde (50% paraformaldehyde) and the reaction was stirred at room temperature for 1.5 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 75 mg of the product. LC-MS (METHOD 5): R _t = 4.03 min; MS (ESIpos): m/z = 1181 [M+H] ⁺ intermediate 109

(3R)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(benzyloxy)carbonyl]amino}-4-oxobutyl acid tributyl ester

To a solution of N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (769 mg, 1.32 mmol) in DMF (76 ml) were added (2R)-2-{[(benzyloxy)carbonyl]amino}-4-tributyloxy-4-oxobutanoic acid (554 mg, 1.71 mmol), HATU (751 mg, 1.98 mmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (460 µl, 2.6 mmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 820 mg of the product. LC-MS (Method 1): R _t = 1.29 min; MS (ESIpos): m/z = 889 [M+H] ⁺ intermediate 110

(3R)-3-amino-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-4-oxobutyl acid tributyl ester

To a solution of (3R)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(benzyloxy)carbonyl]amino}-4-oxobutanoic acid tributyl ester (820 mg, 922 µmol) in methanol/DCM (60 ml, 1:1) was added 10% palladium/activated carbon (40 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 685 mg of the title product. LC-MS (Method 1): R _t = 0.85 min; MS (ESIpos): m/z = 755 [M+H] ⁺ intermediate 111

(3R)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-4-oxobutyl acid tributyl ester

To a solution of (3R)-3-amino-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-4-oxobutyric acid tert-butyl ester (685 mg, 907 µmol) in DMF (52 ml) were added N-[(benzyloxy)carbonyl]-L-alanine (263 mg, 1.18 mmol), HATU (518 mg, 1.36 mmol; CAS-RN:148893-10-1) and N,N -diisopropylethylamine (320 µl, 1.8 mmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 753 mg of the product. LC-MS (Method 1): R _t = 1.25 min; MS (ESIpos): m/z = 960 [M+H] ⁺ intermediate 112

(3R)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-aminopropionyl]amino}-4-oxobutyl acid tributyl ester

To a solution of (3R)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-{[(benzyloxy)carbonyl]amino}propionyl]amino}-4-oxobutanoic acid tributyl ester (753 mg, 784 µmol) in methanol/DCM (50 ml, 1:1) was added 10% palladium/activated carbon (40 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 618 mg of the title product. LC-MS (Method 1): R _t = 0.92 min; MS (ESIpos): m/z = 826 [M+H] ⁺ intermediate 113

(9S,12S,15R)-15-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminocarbonyl}-9-[(t-butyloxycarbonyl)amino]-12-methyl-3,10,13-trioxo-1-phenyl-2-oxa-4,11,14-triazaheptadecan-17-oic acid tributyl ester

To a solution of (3R)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-aminopropionyl]amino}-4-oxobutyric acid tert-butyl ester (150 mg, 182 µmol) in DMF (15 ml) were added N6-[(benzyloxy)carbonyl]-N2-(tert-butyloxycarbonyl)-L-lysine (72.5 mg, 191 µmol), HATU (138 mg, 363 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (95 µl, 540 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 15 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 141 mg of the product. LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 1188 [M+H] ⁺ intermediate 114

(6S,9S,12R)-12-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-6-(4-aminobutyl)-2,2,9-trimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatetradec-14-oic acid tributyl ester

To a solution of (9S,12S,15R)-15-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminocarbonyl}-9-[(t-butyloxycarbonyl)amino]-12-methyl-3,10,13-trioxo-1-phenyl-2-oxazolidin-4,11,14-triazaheptadecan-17-oic acid tributyl ester (141 mg, 119 µmol) in methanol/DCM (30 ml, 1:1) was added 10% palladium/activated carbon (20 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 131 mg of the title product. LC-MS (Method 1): R _t = 0.94 min; MS (ESIpos): m/z = 1054 [M+H] ⁺ intermediate 115

(2R)-2-{[(6S,9S,12R,15S,27S,30S,33R,36S,43R)-9,36-bis(2-amino-2-oxoethyl)-43-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl) 27-[(t-butyloxycarbonyl)amino]ethyl}-33-(2-t-butyloxy-2-oxoethyl)-42-(hydroxyacetyl)-12,15,30,44,44-pentamethyl-5,8,11,14,17,21,28,31,34,37-decanoyl-4,7,10,13,16,22,29,32,35,38,42-undecanopentatradecanoyl]amino}glutaric acid

(6S,9S,12R)-12-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-6-(4-aminobutyl)-2,2,9-trimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatetradec-14-oic acid tributyl ester (69.6 mg, 66.0 To a solution of 2-[(4-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino]-D-propylamino]-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (78.0 mg, 66.0 μmol) in DMF (8.0 ml) was added N,N -diisopropylethylamine (58 μl, 330 μmol). µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 2 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 79 mg of the product. LC-MS (Method 1): R _t = 1.24 min; MS (ESIneg): m/z = 1058 [M-2H] ² - Intermediate 116

(2R,5S,8S,20S,23R,26S,29S,36R)-8-amino-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-26-(2-amino-2-oxoethyl) 2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,20,23-trimethyl-4,7,14,18,21,24,27,30,34-nonaoxo-3,6,13,19,22,25,28,31,35-nonaazatrioctadecane-1,36,38-tricarboxylic acid

(2R)-2-{[(6S,9S,12R,15S,27S,30S,33R,36S,43R)-9,36-bis(2-amino-2-oxoethyl)-43-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl) [0366] Amino]ethyl}-27-[(t-butyloxycarbonyl)amino]-33-(2-t-butyloxy-2-oxoethyl)-42-(hydroxyacetyl)-12,15,30,44,44-pentamethyl-5,8,11,14,17,21,28,31,34,37-decanoyl-4,7,10,13,16,22,29,32,35,38,42-undezapentatetradecanoyl]amino}pentanedioic acid (79.0 mg, 37.3 µmol) was dissolved in 2,2,2-trifluoroethanol (10 ml). Zinc chloride (40.6 mg, 298 µmol) was added and the reaction was stirred at 50°C for 8 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (87.1 mg, 298 µmol; CAS-RN: [60-00-4]) was added and the mixture was diluted with water/acetonitrile. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 54 mg of product. LC-MS (Method 5): R _t = 4.46 min; MS (ESIpos): m/z = 982 [M+2H] ²⁺ Intermediate 117

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(tert-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylamino-D-glutamine diphenylmethyl ester

To a solution of N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (200 mg, 194 µmol) and N2-(tert-butyloxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidin-1-yl ester (70.1 mg, 213 µmol; CAS-RN:42002-18-6) in DMF (12 ml) was added N,N -diisopropylethylamine (100 µl, 580 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 4.5 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 183 mg of the product. LC-MS (Method 1): R _t = 1.34 min; MS (ESIpos): m/z = 1108 [M+H] ⁺ intermediate 118

N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine diphenylmethyl ester

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N2-(t-butyloxycarbonyl)-L-asparagine]amino}butyryl]-β-propylaminoyl-D-glutamine diphenylmethyl ester (350 mg, 316 µmol) was dissolved in 2,2,2-trifluoroethanol (30 ml). Zinc chloride (258 mg, 1.89 mmol) was added and the reaction was stirred at 50°C for 3 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (554 mg, 1.89 mmol; CAS-RN: 60-00-4) was added, followed by 5 ml of water/0.1% TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 284 mg of product. LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 1008 [M+H] ⁺ intermediate 119

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5-azaeicosan-20-yl)-L-asparagine]amino}butyryl]-β-propylamino-D-glutamine diphenylmethyl ester

To a solution of N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (23.0 mg, 20.5 µmol), 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosanoic acid (8.99 mg, 24.6 µmol) and HATU (8.57 mg, 22.5 µmol; CAS-RN: 148893-10-1) in DMF (2.3 ml) was added N,N -diisopropylethylamine (11 µl, 61 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 5 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 21 mg of the product. LC-MS (Method 1): R _t = 1.31 min; MS (ESIpos): m/z = 1356 [M+H] ⁺ intermediate 120

N-{(2S)-2-{[N ² -(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)-L-asparagine]amino}-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine diphenylmethyl ester

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-{[N ² -(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5-azaeicosan-20-yl)-L-asparagine]amino}butyryl]-β-propylaminoyl-D-glutamine diphenylmethyl ester (21.0 mg, 15.5 µmol) was dissolved in 2,2,2-trifluoroethanol (8 ml). Zinc chloride (12.7 mg, 93.0 µmol) was added and the reaction was stirred at 50°C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (27.2 mg, 93.0 µmol; CAS-RN: 60-00-4) was added, followed by 4 ml of water/acetonitrile (1:1) and TFA (20 µl), and the mixture was stirred for 10 minutes. The resulting solid was filtered off, the mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 17 mg of the product. LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m/z = 1255 [M+H] ⁺ intermediate 121

(4S)-5-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid tributyl ester

To a solution of N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (50.0 mg, 85.7 µmol) and 5-tert-butyl 1-(2,5-dioxopyrrolidin-1-yl) N-[(benzyloxy)carbonyl]-L-glutamine (44.7 mg, 103 µmol) in DMF (4.0 ml) was added N,N -diisopropylethylamine (45 µl, 260 µmol; CAS-RN:7087-68-5) and the reaction was stirred at room temperature for 12 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 51 mg of product. LC-MS (Method 1): R _t = 1.29 min; MS (ESIpos): m/z = 903 [M+H] ⁺ intermediate 122

N-[(Benzyloxy)carbonyl]-L-α-aminoacyl-N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-asparagine

(4S)-5-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid tributyl ester (51.0 mg, 56.5 µmol) was dissolved in 2,2,2-trifluoroethanol (6 ml). Zinc chloride (46.2 mg, 339 µmol) was added and the reaction was stirred at 50°C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (99.0 mg, 339 µmol; CAS-RN: [60-00-4]) was added, followed by 4 ml of water and TFA (50 µl), and the mixture was stirred for 10 minutes. The resulting solid was filtered off, the filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 45 mg of the product. LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 847 [M+H] ⁺ intermediate 123

(2R)-2-{[(6S,9S,30S,33S,40R)-9,33-bis(2-amino-2-oxoethyl)-40-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethyl Propyl}(hydroxyacetyl)amino]ethyl}-30-{[(benzyloxy)carbonyl]amino}-39-(hydroxyacetyl)-41,41-dimethyl-5,8,11,27,31,34-hexaoxo-14,17,20,23-tetraoxa-4,7,10,26,32,35,39-heptaazatetradecanoyl]amino}diphenylmethyl glutarate

N-{(2S)-2-{[N ² -(15-amino-4,7,10,13-tetraoxopentadec-1-yl)-L-asparaginyl]amino}-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylamino-D-glutamine diphenylmethyl ester (17.0 mg, 12.4 µmol) and N-[(benzyloxy)carbonyl]-L-α-glutamido-N ¹ To a solution of 3-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (10.6 mg, 12.4 µmol) in DMF (4.0 ml) were added HATU (5.66 mg, 14.9 µmol; CAS-RN:148893-10-1) and N,N-diisopropylethylamine (6.5 µl, 37 µmol; CAS-RN:7087-68-5), and the reaction was stirred at room temperature for 5 min. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 20 mg of the product. LC-MS (Method 1): R _t = 1.45 min; MS (ESIpos): m/z = 1042 [M+2H] ²⁺ Intermediate 124

(2R)-2-{[(6S,9S,30S,33S,40R)-30-amino-9,33-bis(2-amino-2-oxoethyl)-40-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H- Pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-39-(hydroxyacetyl)-41,41-dimethyl-5,8,11,27,31,34-hexaoxy-14,17,20,23-tetraoxa-4,7,10,26,32,35,39-heptaazatetradecanoyl]amino}glutaric acid

(2R)-2-{[(6S,9S,30S,33S,40R)-9,33-bis(2-amino-2-oxoethyl)-40-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl To a solution of diphenylmethyl pentanoate (20.0 mg, 99% purity, 9.47 µmol) in methanol/DCM (10 ml, 1:1) was added 10% palladium/activated carbon (2 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 11 mg of the title product. LC -MS (Method 1): R _t = 1.02 min; MS (ESIneg): m/z = 1767 [MH] ^{-Intermediate} 125

(3R,10S,13S,16S,19S)-10-(2-amino-2-oxoethyl)-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-19-[(t-butyloxycarbonyl)amino]-13-(2-t-butyloxy-2-oxoethyl)-4-(hydroxyacetyl)-2,2,16-trimethyl-9,12,15,18-tetraoxo-4,8,11,14,17-pentaazadocosan-22-oic acid benzyl ester

To a solution of (3S)-4-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]amino}-3-{[(2S)-2-aminopropionyl]amino}-4-oxobutyric acid tributyl ester (30.0 mg, 36.3 µmol) in DMF (5.0 ml) were added (2S)-5-(benzyloxy)-2-[(t-butyloxycarbonyl)amino]-5-oxopentanoic acid (17.2 mg, 50.8 µmol), HATU (27.6 mg, 72.6 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (19 µl, 110 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 38 mg of the product. LC-MS (Method 1): R _t = 1.32 min; MS (ESIpos): m/z = 1146 [M+H] ⁺ intermediate 126

(3R,10S,13S,16S,19S)-10-(2-amino-2-oxoethyl)-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-19-[(t-butyloxycarbonyl)amino]-13-(2-t-butyloxy-2-oxoethyl)-4-(hydroxyacetyl)-2,2,16-trimethyl-9,12,15,18-tetraoxo-4,8,11,14,17-pentazadocosan-22-oic acid

To a solution of (3R,10S,13S,16S,19S)-10-(2-amino-2-oxoethyl)-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-19-[(t-butyloxycarbonyl)amino]-13-(2-t-butyloxy-2-oxoethyl)-4-(hydroxyacetyl)-2,2,16-trimethyl-9,12,15,18-tetraoxo-4,8,11,14,17-pentazadocosan-22-oic acid benzyl ester (38.0 mg, 33.2 µmol) in methanol/DCM (13 ml, 1:1) was added 10% palladium/activated carbon (8 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized. The residue was purified by preparative HPLC to give 12 mg of the title product. LC-MS (Method 1): R _t = 1.17 min; MS (ESIpos): m/z = 1055 [M+H] ⁺ intermediate 127

(2R)-2-{[(6S,9S,12S,15S)-12-(2-amino-2-oxoethyl)-15-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-2,2,6,9-tetramethyl-4,7,10,13,16,20-hexaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-20-yl]amino}diphenylmethyl glutarate

To a solution of N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (100 mg, 89.1 µmol) in DMF (10 ml) were added N-(tert-butyloxycarbonyl)-L-propylaminoyl-L-alanine (27.8 mg, 107 µmol), HATU (40.7 mg, 107 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (62 µl, 360 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 96 mg of the product. LC-MS (Method 1): R _t = 1.30 min; MS (ESIpos): m/z = 1250 [M+H] ⁺ intermediate 128

(2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12-methyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}diphenylmethyl glutarate

(2R)-2-{[(6S,9S,12S,15S)-12-(2-amino-2-oxoethyl)-15-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-2,2,6,9-tetramethyl-4,7,10,13,16,20-hexaneoxo-3-oxa-5,8,11,14,17-pentaazaicostan-20-yl]amino}pentanedioic acid dibenzhydryl ester (96.0 mg, 76.8 µmol) was dissolved in 2,2,2-trifluoroethanol (5 ml). Zinc chloride (62.8 mg, 461 µmol) was added and the reaction was stirred at 50 °C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (135 mg, 461 µmol; CAS-RN: [60-00-4]) was added followed by 2 ml of water/0.1% TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 69 mg of product. LC-MS (Method 1): R _t = 1.01 min; MS (ESIpos): m/z = 1151 [M+H] ⁺ intermediate 129

(2S,5S,8S,13S,16S,19S,22S,29R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-19-(2-amino-2-oxoethyl)-22-{2-[{(1R) -1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-8-[(t-butyloxycarbonyl)amino]-5,13,16-trimethyl-4,7,11,14,17,20,23,27-octanoyloxy-3,6,12,15,18,21,24,28-octaazatriacontane-1,29,31-tricarboxylic acid 29,31-ditolyl 1-tributyl ester

To (3R,10S,13S,16S,19S)-10-(2-amino-2-oxoethyl)-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-19-[(t-butyloxycarbonyl)amino]-13-(2-t-butyloxy-2-oxoethyl)-4-(hydroxyacetyl)-2,2,16-trimethyl-9,12,15,18-tetraoxo-4,8,11,14,17-pentazadocosan-22-oic acid (32.4 mg, 27.7 µmol) in DMF (8.1 To a solution of 10 μl (20 μl) in 4% paraformaldehyde (20 μl), (2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12-methyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}pentanedioic acid diphenylmethyl ester (35.0 mg, 27.7 μmol), HATU (21.1 mg, 55.4 μmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (14 μl, 83 μmol) were added. µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 30 min. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 40 mg of the product. LC-MS (Method 1): R _t = 1.51 min; MS (ESIpos): m/z = 1094 [M+2H] ²⁺ Intermediate 130

(6R,13S,16S,19S,22S,27S,30S,33S)-27-amino-33-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-16-(2-amino-2-oxoethyl)-13- {2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-6-[(benzyloxy)carbonyl]-19,22,30-trimethyl-3,8,12,15,18,21,24,28,31-nonaoxy-1-phenyl-2-oxa-7,11,14,17,20,23,29,32-octaazapentatriacontanoic acid

(2S,5S,8S,13S,16S,19S,22S,29R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminocarbonyl}-19-(2-amino-2-oxoethyl)-22-{2-[{(1R)-1 1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-8-[(t-butyloxycarbonyl)amino]-5,13,16-trimethyl-4,7,11,14,17,20,23,27-octaoxy-3,6,12,15,18,21,24,28-octaazatriacontane-1,29,31-tricarboxylic acid 29,31-diphenylmethyl ester 1-t-butyl ester (39.6 mg, 18.1 µmol) was dissolved in 2,2,2-trifluoroethanol (4 ml). Zinc chloride (14.8 mg, 109 µmol) was added and the reaction was stirred at 50°C for 2 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (31.7 mg, 109 µmol; CAS-RN: 60-00-4) was added followed by 2 ml of water/0.1% TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 25 mg of product. LC-MS (Method 1): R _t = 1.27 min; MS (ESIpos): m/z = 1016 [M+2H] ²⁺ Intermediate 131

(2S,5S,8S,13S,16S,19S,22S,29R)-8-amino-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminocarbonyl}-19-(2-amino-2- 2-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,13,16-trimethyl-4,7,11,14,17,20,23,27-octanoyloxy-3,6,12,15,18,21,24,28-octazatrionedecane-1,29,31-tricarboxylic acid

(6R,13S,16S,19S,22S,27S,30S,33S)-27-amino-33-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl}-16-(2-amino-2-oxoethyl)-13-{2 To a solution of -[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-6-[(benzyloxy)carbonyl]-19,22,30-trimethyl-3,8,12,15,18,21,24,28,31-nonaoxy-1-phenyl-2-oxa-7,11,14,17,20,23,29,32-octaazapentatriacontanoic acid (25.0 mg, 11.7 µmol) in methanol/DCM (12 ml, 1:1) was added 10% palladium/activated carbon (2 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 20 mg of the title product. LC-MS (Method 1): R _t = 1.06 min; MS (ESIneg): m/z = 923 [M-2H] ^2- Intermediate 132

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl 2-{[(benzyloxy)carbonyl]amino}-16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester

To a solution of (2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12-methyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}pentanedioic acid diphenylmethyl ester (20.0 mg, 15.8 µmol) in DMF (4.4 ml) were added N-[(benzyloxy)carbonyl]-L-glutamine (2.22 mg, 7.91 µmol), HATU (9.02 mg, 23.7 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (6.9 µl, 40 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 2.5 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 12 mg of the product. LC-MS (Method 1): R _t = 1.53 min; MS (ESIpos): m/z = 1273 [M+2H] ²⁺ Intermediate 133

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-22-amino-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethyl 16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecaazapentatetradecan-1,3,43,45-tetracarboxylic acid

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl} To a solution of 2,2-{[(benzyloxy)carbonyl]amino}-16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecaazapentatetradecan-1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester (12.0 mg, 4.71 µmol) in methanol/DCM (8 ml, 1:1) was added 10% palladium/activated carbon (9 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 10 mg of the title product. LC-MS (Method 1): R _t = 0.94 min; MS (ESIneg): m/z = 1024 [M-2H] ^2- Intermediate 134

(3R,10S,13S,16S,21S,24S,31R)-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16-{[(benzyloxy)carbonyl]amino}-5,9,12,15,19,22,25,29-octanooxo-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid tetraphenylmethyl ester

To a solution of N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (70.0 mg, 62.4 µmol) in DMF (10 ml) was added a solution of N-[(benzyloxy)carbonyl]-L-glutamine (8.77 mg, 31.2 µmol) in DMF, HATU (35.6 mg, 93.6 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (27 µl, 160 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 1.5 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 50 mg of the product. LC-MS (Method 1): R _t = 1.58 min; MS (ESIpos): m/z = 1131 [M+2H] ²⁺ Intermediate 135

(3R,10S,13S,16S,21S,24S,31R)-16-amino-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,9,12,15,19,22,25,29-octanooxo-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid

To (3R,10S,13S,16S,21S,24S,31R)-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16-{[(benzyloxy)carbonyl]amino}-5,9,12,15,19,22,25,29-octaoxo-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid tetraphenylmethyl ester (50.0 mg, 22.1 µmol) in methanol/DCM (20 10% palladium/activated carbon (18 mg) was added to a solution of 4-nitropropene (2-nitropropene) in 4% paraffin (1:1, 4-nitropropene). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 39 mg of the title product. LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m/z = 1767 [M+H] ⁺ intermediate 136

(3R,10S,13S,18S,21S,26S,29S,36R)-13,26-bis(2-amino-2-oxoethyl)-10,29-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-21-{[(benzyloxy)carbonyl]amino}-5,9,12,15,20,24,27,30,34-nonaoxo-4,8,11,14,19,25,28,31,35-nonaazatrioctadecane-1,3,18,36,38-pentacarboxylic acid pentaphenylmethyl ester

To a solution of N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine diphenylmethyl ester (40.0 mg, 35.6 µmol) and (4S)-4-{[(benzyloxy)carbonyl]amino}-5-{[(2S)-1-(benzyloxy)-4-carboxy-1-oxobutyl-2-yl]amino}-5-oxopentanoic acid (8.92 mg, 17.8 µmol) in DMF (5.0 ml) was added HATU (17.6 mg, 46.3 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (19 µl, 110 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 10 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 29 mg of the product. LC-MS (Method 1): R _t = 1.60 min; MS (ESIpos): m/z = 1241 [M+2H] ²⁺ Intermediate 137

N-[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}amino)-1,4-dioxobutyl-2-yl]-L-glutamido-L-γ-glutamido-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

(3R,10S,13S,18S,21S,26S,29S,36R)-13,26-bis(2-amino-2-oxoethyl)-10,29-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-21-{[(benzyloxy)carbonyl]amino}-5,9,12,15,20,24,27,30,34-nonaoxo-4,8,11,14,19,25,28,31,35-nonaazatrioctadecane-1,3,18,36,38-pentacarboxylic acid pentaphenylmethyl ester (28.5 To a solution of 147 mg, 11.5 µmol) in methanol (15 ml) was added 10% palladium/activated carbon (3 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 21 mg of the title product. LC-MS (Method 1): R _t = 1.00 min; MS (ESIpos): m/z = 1896 [M+H] ⁺ intermediate 138

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl }-22-[(tributyloxycarbonyl)amino]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester

To a solution of N-(tert-butyloxycarbonyl)-L-glutamine (2.71 mg, 11.0 µmol) and (2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12,13-dimethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}pentanedioic acid diphenylmethyl ester (28.0 mg, 21.9 µmol) in DMF (2.0 ml) was added HATU (9.99 mg, 26.3 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (7.6 µl, 44 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature for 10 min. Water/0.1% TFA was added, the resulting solid was filtered off, and the filtrate was purified by preparative HPLC to give 20 mg of the product. Intermediate 139

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-22-amino-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}( [Hydroxyacetyl]amino]ethyl]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanepentatetradecane-1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}- 22-[(tributyloxycarbonyl)amino]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecahydropentatetradecane-1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester (20.3 mg, 7.99 µmol) was dissolved in 2,2,2-trifluoroethanol (3 ml). Zinc chloride (6.54 mg, 48.0 µmol) was added and the reaction was stirred at 50°C. After stirring for 3 hours, zinc chloride (6.54 mg, 48.0 µmol) was added, and the reaction was stirred at 50 °C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (28.0 mg, 95.9 µmol; CAS-RN: [60-00-4]) was added, and the mixture was stirred for 10 min. Water/0.1% TFA was added, and the solid was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 34 mg of product. LC-MS (Method 5): R _t = 5.75 min; MS (ESIpos): m/z = 1015 [M+H] ²⁺ Intermediate 140

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-[(2,2-dimethyl-4,20-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl} [3,8,11,14,17-pentaoxadiazine-5-azaeicosane-20-yl)amino]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecanoic acid tetraphenylmethyl ester

To (3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-22-amino-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxy [acetyl)amino]ethyl]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanepentatetradecane-1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester (16.5 To a solution of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosane-20-oic acid (2.83 mg, 7.75 µmol) in DMF (2.0 ml) were added HATU (2.95 mg, 7.75 µmol; CAS-RN: 148893-10-1) and N,N -diisopropylethylamine (4.5 µl, 26 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 10 min. The mixture was filtered, and the filtrate was purified by preparative HPLC to give 11 mg of the product. LC-MS (Method 5): R _t = 6.98 min; MS (ESIpos): m/z = 1394 [M+2H] ²⁺ Intermediate 141

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-22-[(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)amino]-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2 1,3,43,45-tetracarboxylic acid tetraphenylmethyl ester

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-[(2,2-dimethyl-4,20-dioxoethyl) [0136] Tetraphenylmethyl-1,3,43,45-tetracarboxylate (10.8 mg, 3.87 µmol) was dissolved in 2,2,2-trifluoroethanol (2 ml). Zinc chloride (3.17 mg, 23.2 µmol) was added and the reaction was stirred at 50 °C. After stirring for 1.5 hours, zinc chloride (3.17 mg, 23.2 µmol l) was added, and the reaction was stirred at 50 °C for 1.5 hours. Zinc chloride (3.17 mg, 23.2 µmol) was added again, and the reaction was stirred at 50 °C until completion. Ethylenediamine-N,N,N',N'-tetraacetic acid (20.4 mg, 69.7 µmol; CAS-RN: [60-00-4]) was added, and the mixture was stirred for 10 min. Water/0.1% TFA was added, and the solid was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 6 mg of product. LC-MS (Method 5): R _t = 5.67 min; MS (ESIpos): m/z = 1344 [M+2H] ²⁺ Intermediate 142

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-22-[(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)amino]-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole 1,3,43,45-tetradecanoic acid

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-22-[(15-amino-4,7,10,13-tetrahydropentadecan-1-yl)amino]-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl To a solution of 1,3,43,45-tetracarboxylic acid tetraphenylmethyl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecazapentatetradecan-1,3,43,45-tetracarboxylate (23.0 mg, 8.21 µmol) in ethyl acetate/ethanol (2 ml, 1:1) was added 10% palladium/activated carbon. The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 3 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 17 mg of the title product. LC-MS (Method 5): R _t = 4.23 min; MS (ESIpos): m/z = 1163 [M+2H] ²⁺ Intermediate 143

(2R)-2-{[(5S,8S,11S,14S)-11-(2-amino-2-oxoethyl)-14-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,7,8-trimethyl-3,6,9,12,15,19-hexaoxo-1-phenyl-2-oxo-4,7,10,13,16-pentazanonadecan-19-yl]amino}pentanedioic acid di-tributyl ester

To a solution of N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (321 mg, 388 µmol) in DMF (20 ml) were added N-[(benzyloxy)carbonyl]-L-propylaminoyl-N-methyl-L-propylaminoyl-L-aspartic acid (254 mg, 99% purity, 466 µmol), HATU (221 mg, 583 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (200 µl, 1.2 mmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 5 min. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 363 mg of the product. LC-MS (Method 2): R _t = 2.49 min; MS (ESIpos): m/z = 1230 [M+H] ⁺ Intermediate 144 (2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12,13-dimethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}pentanedioic acid di-tributyl ester

To a solution of (2R)-2-{[(5S,8S,11S,14S)-11-(2-amino-2-oxoethyl)-14-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,7,8-trimethyl-3,6,9,12,15,19-hexaoxo-1-phenyl-2-oxazolidin-4,7,10,13,16-pentazanonadecan-19-yl]amino}pentanedioic acid di-tributyl ester (363 mg, 295 µmol) in ethanol (30 ml) was added 10% palladium/activated carbon (30 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 2 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 206 mg of the product. LC-MS (Method 1): R _t = 1.06 min; MS (ESIpos): m/z = 1096 [M+H] ⁺ intermediate 145

(3R,10S,13S,16S,19S,22R,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22 -[3-(2-bromoacetamido)propionamido]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid tetra-tributyl ester

N-(Bromoacetyl)-β-propylaminoyl-D-glutamine (2.66 mg, 7.30 µmol) and (2R)-2-{[(6S,9S,12S,15S)-15-amino-9-(2-amino-2-oxoethyl)-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-12,13-dimethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazahexadec-1-yl]amino}pentanedioic acid di-tributyl ester (20.0 mg, 18.2 µmol) in DMF (5.0 HATU (6.94 mg, 18.2 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (6.4 µl, 36 µmol; CAS-RN: 7087-68-5) were added to a solution of 1% 4-nitropropanediol (2% 4-nitropropanediol) in 4% 4-nitropropanediol (1% 4-nitropropanediol) and the reaction was stirred at room temperature for 10 min. The mixture was concentrated to dryness under reduced pressure and the residue was combined with 2 mg of another batch synthesized in this way. The mixture was purified by preparative HPLC to give 12 mg of the product.

LC-MS (Method 4): R _t = 7.73 min; MS (ESIpos): m/z = 1270 [M+2Na] ²⁺ Intermediate 146

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-38-{[(benzyloxy)carbonyl]amino}-16 ,17,19,59,61,62-hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoylhexadecane-1,3,75,77-tetracarboxylic acid tetra-tributyl ester

To (2S,5S,8S,27S,48S,51S,54S)-2,54-bis(2-amino-2-oxoethyl)-27-{[(benzyloxy)carbonyl]amino}-5,6,8,48,50,51-hexamethyl-4,7,10,26,30,46,49,52-octaoxo-13,16,19,22,34,37,40,43-octaoxa-3,6,9,25,31,47,50,53-octaazapentacontan-1,55-dioic acid (13.4 mg, 10.2 To a solution of 2-[(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (16.8 mg, 20.4 µmol) in DMF (1.2 ml) were added HATU (9.29 mg, 24.4 µmol; CAS-RN:148893-10-1) and N,N-diisopropylethylamine (11 µl, 61 µmol; CAS-RN:7087-68-5), and the reactants were stirred at room temperature. After stirring for 20 min, N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (16.8 mg, 20.4 µmol), HATU (9.29 mg, 24.4 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (11 µl, 61 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred until completion. Water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 4 mg of product. LC-MS (Method 5): R _t = 6.62 min; MS (ESIpos): m/z = 1466 [M+2H] ²⁺ Intermediate 147

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-38-{[(benzyloxy)carbonyl]amino} -16,17,19,59,61,62-hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoyl-1,3,75,77-tetradecanoic acid

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-38-{[(benzyloxy)carbonyl]amino}-16,1 7,19,59,61,62-Hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoylheptadecane-1,3,75,77-tetracarboxylic acid tetra-tributyl ester (4.10 mg, 1.30 µmol) was dissolved in 2,2,2-trifluoroethanol (2 ml). Zinc chloride (1.06 mg, 7.78 µmol) was added and the reaction was stirred at 50 °C. After 1 hour, more zinc chloride (1.06 mg, 7.78 µmol) was added, and the reaction was stirred at 50 °C for another hour. This procedure was repeated 4 times until the reaction was complete. Ethylenediamine-N,N,N',N'-tetraacetic acid (2.27 mg, 7.78 µmol; CAS-RN: 60-00-4) was added, followed by water/0.1% TFA, and the mixture was stirred for several minutes. The mixture was filtered, and the filtrate was purified by preparative HPLC to give 2.2 mg of the product. Intermediate 148

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-38-amino-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16,17 ,19,59,61,62-hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoyl-1,3,75,77-tetradecanoic acid

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-38-{[(benzyloxy)carbonyl]amino}-16, To a solution of 17,19,59,61,62-hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoylheptadecane-1,3,75,77-tetracarboxylic acid-trifluoroacetic acid (1/2) (2.20 mg, 0.749 µmol) in ethanol (5 ml) was added 10% palladium/activated carbon (1 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 3 hours. The solid was filtered off, the residue was washed several times with ethanol, and the filtrate was concentrated under reduced pressure to obtain 2 mg of crude product. Intermediate 149

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-{[6-(2,5-difluorophenyl)-2,5-difluorophenyl] [(1H-pyrrol-1-yl)hexanoyl]amino]-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid tetra-tributyl ester]

(2S,5S,8S,13S,16S)-8-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-2,16-dimethyl-4,7,11,14-tetraoxo-5,13-di(propan-2-yl)-3,6,12,15-tetraazaheptadecan-1,17-dioic acid (10.0 mg, 14.7 To a solution of 2-[(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (24.3 mg, 29.4 μmol) in DMF (1.5 ml) were added HATU (13.4 mg, 35.3 μmol; CAS-RN:148893-10-1) and N,N-diisopropylethylamine (10 μl, 59 μmol; CAS-RN:7087-68-5), and the reaction was stirred at room temperature for 10 min. Ethyl acetate was added to the mixture, and the mixture was washed with saturated ammonium chloride solution (3 times), saturated sodium bicarbonate solution (3 times) and brine (20 ml) (3 times). The organic phase was dried over magnesium sulfate, filtered and evaporated to dryness to give 32 mg of crude product. LC-MS (Method 4): R _t = 8.10 min; (ESIpos): m/z = 1171 [M+2Na] ²⁺ Intermediate 150

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-({N-[(benzyloxy)carbonyl]-L-carboxamidoyl-L-propylaminoyl}amino)butyryl]-β-propylaminoyl-D-glutamine di-tributyl ester

To a solution of N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine di-tert-butyl ester (500 mg, 605 µmol) and N-[(benzyloxy)carbonyl]-L-glutamidoyl-L-alanine (234 mg, 726 µmol) in DMF (10 ml) were added HATU (276 mg, 726 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (320 µl, 1.8 mmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 10 min. Water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 564 mg of product. LC-MS (Method 1): R _t = 1.43 min; MS (ESIpos): m/z = 1130 [M+H] ⁺ intermediate 151

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-[(L-aminopropyl-L-propylaminopropyl)amino]butyryl}-β-propylaminopropyl-D-glutamine di-tributyl ester

To a solution of N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-({N-[(benzyloxy)carbonyl]-L-hydroxyamido-L-propylamido}amino)butyryl]-β-propylamido-D-glutamine di-tert-butyl ester (564 mg, 499 µmol) in ethyl acetate/ethanol (60 ml, 1:1) was added 10% palladium/activated carbon (56 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 4 hours. The solid was filtered off, washed with ethyl acetate/ethanol, and the filtrate was concentrated under reduced pressure to give 502 mg of the title product. LC-MS (Method 2): R _t = 2.02 min; MS (ESIpos): m/z = 996 [M+H] ⁺ intermediate 152

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-{[(benzyloxy)carbonyl]amino}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid tetra-tributyl ester

To a solution of N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-[(L-hydroxyamidoyl-L-propylamidoyl)amino]butyryl}-β-propylamidoyl-D-glutamine di-tert-butyl ester (150 mg, 151 µmol) in DMF (10 ml) were added N-[(benzyloxy)carbonyl]-L-glutamine (21.2 mg, 75.3 µmol), HATU (85.9 mg, 226 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (66 µl, 380 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 30 min. Water/0.1% TFA was added, the mixture was filtered and the filtrate was purified by preparative HPLC to give 121 mg of product. LC-MS (Method 4): R _t = 8.37 min; MS (ESIpos): m/z = 1141 [M+2Na] ²⁺ Intermediate 153

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-19-amino-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid tetra-tributyl ester

To (3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-{[(benzyloxy) To a solution of tetra-tributyl]-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decaoxy-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decaazanonatriacontane-1,3,37,39-tetracarboxylate (122 mg, 54.3 µmol) in methanol/DCM (8 ml, 1:1) was added 10% palladium/activated carbon (12 mg). The mixture was hydrogenated at room temperature and under standard hydrogen pressure for 12 h. The solid was filtered off and the residue was washed with methanol/DCM. The filtrate was concentrated under reduced pressure. The remaining residue was dissolved in acetonitrile/water and lyophilized to give 97 mg of the title product. LC-MS (Method 5): R _t = 6.10 min; MS (ESIpos): m/z = 1052 [M+2H] ²⁺ Intermediate 154

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-[(2,2-dimethyl-4,20-dioxo-3,8,11 [(1,4,17-pentaoxadiazine-5-azaeicosan-20-yl)amino]-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decadioxy-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decadiazadinonatriacontane-1,3,37,39-tetracarboxylic acid tetra-tributyl ester]

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-19-amino-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid tetra-tributyl ester (97.6 mg, 44.0 To a solution of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosane-20-oic acid (19.3 mg, 52.8 µmol) in DMF (5.0 ml) was added HATU (20.1 mg, 52.8 µmol; CAS-RN: 148893-10-1) and N,N -diisopropylethylamine (23 µl, 130 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 20 min. The reaction was quenched by adding water/0.1 TFA (1 ml), and the mixture was purified by preparative HPLC to give 85 mg of the product. LC-MS (Method 5): R _t = 6.90 min; MS (ESIpos): m/z = 1226 [M+2H] ²⁺ Intermediate 155

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-19-[(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)amino]-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2 -Dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-[(2,2-dimethyl-4,20-dioxo-3,8,11,1 Tetra-butyl 4,17-pentaoxa-5-azaeicosan-20-yl)amino]-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decaoxy-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decaazanonatriacontane-1,3,37,39-tetracarboxylate (85.6 mg, 34.9 µmol) was dissolved in 2,2,2-trifluoroethanol (5 ml). Zinc chloride (28.6 mg, 210 µmol) was added and the reaction was stirred at 50°C. After stirring for 2 hours, zinc chloride (28.6 mg, 210 µmol) was added and the reaction was stirred at 50 °C for 2 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (61.2 mg, 210 µmol; CAS-RN: [60-00-4]) was added and the mixture was stirred for 10 minutes before water/TFA (0.1%) was added. The mixture was filtered and the filtrate was purified by preparative HPLC to give 41 mg of product. LC-MS (Method 5): R _t = 4.54 min; MS (ESIpos): m/z = 1064 [M+2H] ²⁺ Intermediate 156

(3R,10S,13S,21S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-21-({2-[(tert-butyloxycarbonyl)amino]ethyl}aminomethyl)-5,9,12,15,19,27,31,34,37,41-decanoyl-4,8,11,14,20,26,32,35,38,42-decazapentatetradecan-1,3,43,45-tetracarboxylic acid

To a solution of N-{2-[(tert-butyloxycarbonyl)amino]ethyl}-L-dihydrocarbylamide (4.56 mg, 11.5 µmol) and N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-[(N2-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-asparagine)amino]butyryl}-β-propylamino-D-glutamine (30.0 mg, 28.9 µmol) in DMF (5.0 ml) was added N,N-diisopropylethylamine (6.0 µl, 35 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 2 days. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 12 mg of the product. Intermediate 157

(3R,10S,13S,21S,33S,36S,43R)-21-[(2-aminoethyl)aminomethyl]-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,9,12,15,19,27,31,34,37,41-decanoyl-4,8,11,14,20,26,32,35,38,42-decazapentatetradecan-1,3,43,45-tetracarboxylic acid

(3R,10S,13S,21S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amine =1,3,43,45-tetracarboxylic acid (12.3 mg, 5.76 µmol) was dissolved in 2,2,2-trifluoroethanol (4 ml). Zinc chloride (4.71 mg, 34.5 µmol) was added and the reaction was stirred at 50 °C for 2.5 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (10.1 mg, 34.5 µmol; CAS-RN: 60-00-4) was added followed by acetonitrile and water. The mixture was filtered and the filtrate was purified by preparative HPLC to give 11 mg of product. LC-MS (Method 1): R _t = 0.93 min; MS (ESIpos): m/z = 1019 [M+2H] ²⁺ A2DC prodrug intermediate 158

(3R,10S,13S,16S,19S,27S,39S,42S,45S,48S,55R)-13,45-bis(2-amino-2-oxoethyl)-10,48-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-55-[2-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl} [(2,5-dihydro-1H-pyrrol-1-yl)acetamido)-16,17,19,39,42-pentamethyl-5,9,12,15,18,21,25,33,37,40,43,46,49,54-tetradecanoyl-4,8,11,14,17,20,26,32,38,41,44,47,50,53-tetradecanoylheptadecane-1,3,27,57-tetracarboxylic acid]

(3R,10S,13S,16S,19S,27S,39S,42S,45S,48S,55R)-13,45-bis(2-amino-2-oxoethyl)-10,48-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-27-(tert-butyloxycarbonyl)-55-[2-( [2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-16,17,19,39,42-pentamethyl-5,9,12,15,18,21,25,33,37,40,43,46,49,54-tetradecanoyl-4,8,11,14,17,20,26,32,38,41,44,47,50,53-tetradecanoylheptadecane-1,3,57-tricarboxylic acid (32.5 mg, 13.2 µmol) was dissolved in 2,2,2-trifluoroethanol (4 ml). Zinc chloride (5.41 mg, 39.7 µmol) was added and the reaction was stirred at 50°C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (11.6 mg, 39.7 µmol; CAS-RN: [60-00-4]) was added, followed by 2 ml of water/0.1% TFA. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 22 mg of product. LC-MS (Method 1): R _t = 1.04 min; MS (ESIpos): m/z = 1200 [M+2H] ²⁺ . Intermediate 159

(3R,10S,13S,21S,33S,36S,39S,42S,49R)-13,39-bis(2-amino-2-oxoethyl)-10,42-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-49-[2- [(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-33,36-dimethyl-5,9,12,15,19,27,31,34,37,40,43,48-dodecyloxy-4,8,11,14,20,26,32,35,38,41,44,47-dodecaazapentadecane-1,3,21,51-tetracarboxylic acid

(3R,10S,13S,21S,33S,36S,39S,42S,49R)-13,39-bis(2-amino-2-oxoethyl)-10,42-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-21-(tert-butyloxycarbonyl)- 49-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-33,36-dimethyl-5,9,12,15,19,27,31,34,37,40,43,48-dodecanoyl-4,8,11,14,20,26,32,35,38,41,44,47-dodecahydro-1,3,51-tricarboxylic acid (14.2 mg, 6.17 µmol) was dissolved in 2,2,2-trifluoroethanol (2 ml). Zinc chloride (5.05 mg, 37.0 µmol) was added and the reaction was stirred at 50°C for 4 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (10.8 mg, 37.0 µmol; CAS-RN: [60-00-4]) was added, followed by 2 ml of water/0.1% TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 8 mg of product. LC-MS (Method 1): R _t = 1.05 min; MS (ESIpos): m/z = 1123 [M+2H] ²⁺ . Intermediate 160

(3R,10S,13S,16S,19S,35S,38S,41S,44S,51R)-13,41-bis(2-amino-2-oxoethyl)-10,44-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-51-[2-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl} [(2,5-dihydro-1H-pyrrol-1-yl)acetamido)-16,19,35,38-tetramethyl-5,9,12,15,18,21,33,36,39,42,45,50-dodecyloxy-24,27,30-trioxa-4,8,11,14,17,20,34,37,40,43,46,49-dodecanotrioxane-1,3,53-tricarboxylic acid]

N-{3-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]propionyl}-L-propylaminoyl-L-propylaminoyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-hydroxybutan-2-yl]-L-aspartamide (18.4 mg, 88% purity, 10.6 µmol) and L-propylamino-L-propylamino-N ¹ To a solution of -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (11.5 mg, 10.6 µmol) in DMF (2.0 ml) was added N,N-diisopropylethylamine (7.4 µl, 42 µmol; CAS-RN:7087-68-5) and the reaction was stirred at room temperature for 12 hours. Water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 12 mg of product. LC-MS (Method 5): R _t = 4.82 min; MS (APCI): m/z = 2261 [M] Intermediate 161

(3R,10S,13S,23S,26S,29S,32S,39R)-13,29-bis(2-amino-2-oxoethyl)-10,32-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-39 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-23,26-dimethyl-5,9,12,15,21,24,27,30,33,38-decanoyl-18-oxa-4,8,11,14,22,25,28,31,34,37-decazaheteradecan-1,3,41-tricarboxylic acid

N-(3-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyloxy}propionyl)-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide (15.0 mg, 10.5 To a solution of 1,4-dihydro-2-nitropropane-2-yl-1H-pyrrol-2-yl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyryl}-β-propylaminoyl-D-glutamine (9.86 mg, 10.5 μmol) in DMF (2.0 ml) was added N,N-diisopropylethylamine (5.5 μl, 31 μmol; CAS-RN:7087-68-5) and the reaction was stirred at room temperature for 12 hours. Water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 9 mg of the product. LC-MS (Method 5): R _t = 4.94 min; MS (ESIpos): m/z = 2031 [M+H] ⁺ intermediate 162

(3R,10S,13S,16S,19S,29S,32S,35S,38S,45R)-13,35-bis(2-amino-2-oxoethyl)-10,38-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-45-[2-(2 [(5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-16,19,29,32-tetramethyl-5,9,12,15,18,21,27,30,33,36,39,44-dodecyloxy-24-oxa-4,8,11,14,17,20,28,31,34,37,40,43-dodecanoatetraheptadecane-1,3,47-tricarboxylic acid]

N-(3-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyloxy}propionyl)-L-propylamino-L-propylamino-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dioxopyrrolidin-1-yl)acetyl]-D-α-glutamyl}amino)ethyl]amino}-1-oxobutyl-2-yl]-L-aspartamide (15.0 mg, 10.5 To a solution of ² -[(4-[(4-[(4-[((2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (11.3 mg, 10.5 µmol) in DMF (2.0 ml) was added N,N -diisopropylethylamine (5.5 µl, 31 µmol; CAS-RN: [7087-68-5]), and the reaction was stirred at room temperature for 12 hours. Water/0.1% TFA (1 ml) was added and the mixture was purified by preparative HPLC to give 15 mg of product. LC-MS (Method 5): R _t = 4.85 min; MS (ESIpos): m/z = 2173 [M+H] ⁺ intermediate 163

(2S,5S,13S,25S,28S,31S,34S,41R)-2-{[(2S)-4-amino-1-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]amino}-1,4-dioxobutyl-2-yl]aminomethyl}-31-(2-amino-2-oxoethyl)-34 -{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-41-[2-(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-5,25,28-trimethyl-4,7,11,19,23,26,29,32,35,40-decaazatris-3,6,12,18,24,27,30,33,36,39-decaazatris-1,13,43-tricarboxylic acid

(3S,6S,14S,26S,29S,32S,35S,42R)-3-{[(2S)-4-amino-1-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[3-(methylamino)-3-oxopropyl]amino}-1-oxobutyl-2-yl]amino}-1,4-dioxobutyl-2-yl]aminomethyl}-32-(2-amino-2-oxoethyl)-35-{2-[{ (1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-14-(t-butyloxycarbonyl)-42-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-6,26,29-trimethyl-5,8,12,20,24,27,30,33,36,41-decaazapentatetradecan-1,45-dioic acid (14.6 mg, 6.16 µmol) was dissolved in 2,2,2-trifluoroethanol (1.8 ml). Zinc chloride (5.04 mg, 37.0 µmol) was added and the reaction was stirred at 50 °C for 2 h. Ethylenediamine-N,N,N',N'-tetraacetic acid (10.8 mg, 37.0 µmol; CAS-RN: [60-00-4]) was added followed by 2 ml of water/0.1% TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 12 mg of product. LC-MS (Method 1): R _t = 1.03 min; MS (ESIpos): m/z = 1157 [M+2H] ²⁺ Intermediate 164

(4R,11S,14S,17S,20S,32S)-14-(2-amino-2-oxoethyl)-32-{[(3R,10S,13S,16S)-10-(2-amino-2-oxoethyl)-3-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-4-(hydroxyacetyl)-2,2,13,16-tetramethyl-9,12,15,18,22-pentaoxo-4,8,11,14,17-pentaazadocosapentazone-22- 2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-4-[2-(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-17,20-dimethyl-5,10,13,16,19,22,26-heptadioxy-6,9,12,15,18,21,27-heptaazatricaria-1,33-dioic acid

To a solution of N-(5-{[(1S)-5-amino-1-carboxypentyl]amino}-5-oxopentanyl)-L-propylamino-L-propylamino-N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (6.00 mg, 5.54 µmol) in DMF (2.0 ml) was added N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-L-propylamino-N1-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-aspartamide (6.00 mg, ^5.54 µmol) -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-{[2-({N-[(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-α-glutamidoyl}amino)ethyl]amino}-1-oxobutan-2-yl]-L-aspartamide (7.15 mg, 5.54 µmol) and N,N-diisopropylethylamine (2.9 µl, 17 µmol; CAS-RN:7087-68-5), and the reaction was stirred at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 6 mg of product. LC-MS (Method 1): R _t = 1.10 min; MS (ESIpos): m/z = 1071 [M+2H] ²⁺ Intermediate 165

(2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-22-(2-amino-2-oxoethyl)-25-{2-[{(1R)-1-[1-benzyl-4-( 2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13-[4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonazolyl-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid

To (2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-13-(4-aminobutyl)-22-(2-amino-2-oxoethyl) 2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonazolyl-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid (5.00 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (1.95 mg, 5.97 µmol) and N,N-diisopropylethylamine (1.2 µl, 7.2 µmol; CAS-RN: 7087-68-5) in DMF (2.0 ml) was added and the reaction was stirred at room temperature for 3 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 1.6 mg of the product. LC-MS (Method 1): R _t = 1.13 min; MS (ESIneg): m/z = 1083 [M-2H] ^2- Intermediate 166

(2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-22-(2-amino-2-oxoethyl)-25-{2-[{(1R)-1-[1-benzyl-4-( 2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-13-(4-{[6-(2,5-dihydro-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonazolyl-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid

To (2S,5S,13S,16S,19S,22S,25S,32R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-13-(4-aminobutyl)-22-(2-amino-2-oxoethyl) 2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,16,18,19-tetramethyl-4,7,11,14,17,20,23,26,30-nonazolyl-3,6,12,15,18,21,24,27,31-nonaazatetratriacontane-1,32,34-tricarboxylic acid (4.40 To a solution of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (810 µg, 2.6 µmol) and N,N-diisopropylethylamine (0.73 µl, 4.2 µmol; CAS-RN: 7087-68-5) in DMF (2.0 ml) was added, and the reaction was stirred at room temperature for 3 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 2.8 mg of the product.

LC-MS (Method 1): R _t = 1.15 min; MS (ESIneg): m/z = 1084 [M-2H] ^2- Intermediate 167

(2R,5S,8S,20S,23R,26S,29S,36R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl}-26-(2-amino-2-oxoethyl)-29-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl} 4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-8-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}amino)-5,20,23-trimethyl-4,7,14,18,21,24,27,30,34-nonaoxo-3,6,13,19,22,25,28,31,35-nonaazatrioctadecane-1,36,38-tricarboxylic acid

(2R,5S,8S,20S,23R,26S,29S,36R)-8-amino-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl}-26-(2-amino-2-oxoethyl) -29-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,20,23-trimethyl-4,7,14,18,21,24,27,30,34-nonaoxo-3,6,13,19,22,25,28,31,35-nonaazatrioctadecane-1,36,38-tricarboxylic acid (9.00 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (2.12 mg, 6.50 µmol) and N,N-diisopropylethylamine (1.5 µl, 8.7 µmol; CAS-RN: 7087-68-5) in DMF (2.5 ml) was added and the reaction was stirred at room temperature for 2 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 8 mg of the product. LC-MS (Method 1): R _t = 1.12 min; MS (ESIneg): m/z = 1086 [M-2H] ^2- Intermediate 168

(2R,5S,8S,20S,23R,26S,29S,36R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl}-26-(2-amino-2-oxoethyl)-29-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl} [6-(2,5-difluorophenyl)-1H-pyrrol-2-yl)-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-8-{[6-(2,5-dihydro-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-5,20,23-trimethyl-4,7,14,18,21,24,27,30,34-nonazolyl-3,6,13,19,22,25,28,31,35-nonaazatrioctadecane-1,36,38-tricarboxylic acid

(2R,5S,8S,20S,23R,26S,29S,36R)-8-amino-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminomethyl}-26-(2-amino-2-oxoethyl) -29-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,20,23-trimethyl-4,7,14,18,21,24,27,30,34-nonaoxo-3,6,13,19,22,25,28,31,35-nonaazatrioctadecane-1,36,38-tricarboxylic acid (54.0 To a solution of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (12.8 mg, 41.6 µmol) in DMF (6.0 ml) was added N,N-diisopropylethylamine (23 µl, 130 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 12 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 46 mg of the product. LC-MS (Method 1): R _t = 1.12 min; MS (ESIneg): m/z = 1077 [M-2H] ^2- Intermediate 169

(2R)-2-{[(6S,9S,30S,33S,40R)-9,33-bis(2-amino-2-oxoethyl)-40-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amine 30-{3-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]propionamido}-39-(hydroxyacetyl)-41,41-dimethyl-5,8,11,27,31,34-hexaoxo-14,17,20,23-tetraoxo-4,7,10,26,32,35,39-heptaazatetradecanoyl]amino}pentanedioic acid

(2R)-2-{[(6S,9S,30S,33S,40R)-30-amino-9,33-bis(2-amino-2-oxoethyl)-40-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-6-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol -2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-39-(hydroxyacetyl)-41,41-dimethyl-5,8,11,27,31,34-hexaoxy-14,17,20,23-tetraoxa-4,7,10,26,32,35,39-heptaazatetradecanoyl]amino}pentanedioic acid (5.00 To a solution of 1-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethyl)-1H-pyrrole-2,5-dione (2.46 mg, 7.93 µmol) in DMF (940 µl) was added N,N-diisopropylethylamine (1.4 µl, 7.9 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 2 hours. TFA was added until pH reached 3 to 4. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 2 mg of the product. LC-MS (Method 4): R _t = 6.25 min; MS (ESIpos): m/z = 1964.9 [M+H]+ Intermediate 170

(2S,5S,8S,13S,16S,19S,22S,29R)-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminoformyl}-19-(2-amino-2-oxoethyl)-22-{2-[{(1R)-1-[1 -Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-8-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)-5,13,16-trimethyl-4,7,11,14,17,20,23,27-octaoxooxy-3,6,12,15,18,21,24,28-octaazatrionedecane-1,29,31-tricarboxylic acid

(2S,5S,8S,13S,16S,19S,22S,29R)-8-amino-2-{[(2S)-4-amino-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1,4-dioxobutyl-2-yl]aminocarbonyl}-19-(2-amino-2-oxobutyl) ethyl)-22-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,13,16-trimethyl-4,7,11,14,17,20,23,27-octanoyloxy-3,6,12,15,18,21,24,28-octaazatriacontane-1,29,31-tricarboxylic acid (20.0 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (9.96 mg, 30.5 µmol) and N,N-diisopropylethylamine (7.1 µl, 41 µmol; CAS-RN: 7087-68-5) in DMF (5.0 ml) was added and the reaction was stirred at room temperature for 4 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 19 mg of the product. LC-MS (Method 1): R _t = 1.14 min; MS (ESIneg): m/z = 1029 [M-2H] ^2- Intermediate 171

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-({5 -[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}amino)-16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecanoyl-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatradecane-1,3,43,45-tetracarboxylic acid

To (3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-22-amino-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropane 16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanepentatetradecane-1,3,43,45-tetracarboxylic acid (5.00 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (1.19 mg, 3.66 µmol) and N,N -diisopropylethylamine (0.85 µl, 4.9 µmol; CAS-RN: 7087-68-5) in DMF (1.4 ml) was added and the reaction was stirred at room temperature for 2 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 3 mg of the product. LC-MS (Method 1): R _t = 1.05 min; MS (ESIneg): m/z = 1029 [M-2H] ^2- Intermediate 172

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-{[6 -(2,5-dihydroxy-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid

To (3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-22-amino-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropane 16,19,27,30-tetramethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid (19.0 To a solution of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (3.57 mg, 11.6 µmol) and N,N -diisopropylethylamine (3.2 µl, 19 µmol; CAS-RN: 7087-68-5) in DMF (7.6 ml) was added and the reaction was stirred at room temperature for 3 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 19 mg of the product. LC-MS (Method 1): R _t = 1.06 min; MS (ESIpos): m/z = 1122 [M+2H] ²⁺ Intermediate 173

(3R,10S,13S,16S,21S,24S,31R)-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl) [amino]ethyl}-16-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-5,9,12,15,19,22,25,29-octaoxo-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid

To (3R,10S,13S,16S,21S,24S,31R)-16-amino-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,9,12,15,19,22,25,29-octaoxo-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid (5.00 mg, 2.83 µmol) in DMF (2.5 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (1.09 mg, 3.54 µmol) and N,N-diisopropylethylamine (0.99 µl, 5.7 µmol; CAS-RN: 7087-68-5) were added to a solution in 4% paraformaldehyde (1% paraformaldehyde) (5% paraformaldehyde) (2% paraformaldehyde) (5% paraformaldehyde) (6% paraformaldehyde) (1% paraformaldehyde) (2% paraformaldehyde) (3% paraformaldehyde) (4% paraformaldehyde) (5% paraformaldehyde) (6% paraformaldehyde) (2% paraformaldehyde) (3% paraformaldehyde) (4% paraformaldehyde) (5% paraformaldehyde) (6% paraformaldehyde) (2% paraformaldehyde) (3% paraformaldehyde) (4% paraformaldehyde) (6% paraformaldehyde) (5% paraformaldehyde) (6% paraformaldehyde) (3% paraformaldehyde) (6% paraformaldehyde) (3% paraformaldehyde) (4% paraformaldehyde) (6% paraformaldehyde) (5% paraformaldehyde) (6% paraformaldehyde) (3% paraformaldehyde) (6% paraformaldehyde) (3% paraformaldehyde) (6% paraformaldehyde) ₍₄ % paraformaldehyde) (6% paraformaldehyde) (6% paraformaldehyde) (3% paraformaldehyde) (6% ^{paraformaldehyde} ) (

(3R,10S,13S,16S,21S,24S,31R)-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)-5,9,12,15,19,22,25,29-octanoyloxy-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid

To (3R,10S,13S,16S,21S,24S,31R)-16-amino-13,21-bis(2-amino-2-oxoethyl)-10,24-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,9,12,15,19,22,25,29-octaoxo-4,8,11,14,20,23,26,30-octaazatricaria-1,3,31,33-tetracarboxylic acid (15.0 mg, 8.49 µmol) in DMF (7.5 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (8.31 mg, 25.5 µmol) and N,N -diisopropylethylamine (4.4 µl, 25 µmol; CAS-RN: 7087-68-5) were added to a solution in 4 ml and the reaction was stirred at room temperature for 4 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 11 mg of the product. LC-MS (Method 1): R _t = 1.12 min; MS (ESIpos): m/z = 1978 [M+H] ⁺ intermediate 175

N-[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}amino)-1,4-dioxobutyl-2-yl]-N ² -{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-glutamidoyl-L-γ-glutamidoyl-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

N-[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}amino)-1,4-dioxobutyl-2-yl]-L-glutamido-L-γ-glutamido-N ¹ To a solution of 1-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (5.00 mg, 2.64 µmol) in DMF (4.0 ml) were added 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (2.15 mg, 6.59 µmol) and N,N-diisopropylethylamine (1.8 µl, 11 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 18 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 2 mg of the product. LC-MS (Method 1): R _t = 1.06 min; MS (ESIneg): m/z = 1052 [M-2H] ² - Intermediate 176

N-[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}amino)-1,4-dioxobutyl-2-yl]-N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-glutamido-L-γ-glutamido-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide

N-[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}amino)-1,4-dioxobutyl-2-yl]-L-glucamido-L-γ-glucamido-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutyl-2-yl}-L-aspartamide (5.00 mg, 2.64 µmol), 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (1000 µg, 4.0 µmol) and N,N-diisopropylethylamine (1.4 µl, 7.9 µmol; CAS-RN: 7087-68-5) in DMF (4.0 ml) was stirred at room temperature for 18 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 2 mg of the product. LC-MS (Method 1): R _t = 1.06 min; MS (ESIneg): m/z = 1015 [M-2H] ^2- . Intermediate 177

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-({21-[(2,5-dioxopyrrolidin-1-yl)- ()oxy]-17,21-dioxo-4,7,10,13-tetraoxa-16-azaheneicosane-1-yl}amino)-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecanoyl-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatradecan-1,3,43,45-tetracarboxylic acid

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-22-[(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)amino]-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2 -yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanepentatetradecane-1,3,43,45-tetracarboxylic acid (5.00 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (1.67 mg, 5.12 µmol) in DMF (500 µl) was added N,N-diisopropylethylamine (1.4 µl, 8.2 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1 hour. Water/0.1% TFA was added and the mixture was purified by preparative HPLC to give 3 mg of the product. Intermediate 178

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-{[22-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-1-yl]amino}-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecanoyl-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-22-[(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)amino]-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2 -yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanepentatetradecane-1,3,43,45-tetracarboxylic acid (5.00 To a solution of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (690 µg, 2.3 µmol) in DMF (500 µl) was added N,N-diisopropylethylamine (1.8 µl, 10 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 3 hours. Water/0.1% TFA was added and the mixture was purified by preparative HPLC to give 3 mg of the product. Intermediate 179

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-{[1-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-yl]amino}-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecanoyl-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid

(3R,10S,13S,16S,19S,22S,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-22-[(15-amino-4,7,10,13-tetraoxopentadecane-1-yl)amino]-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2 -yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanepentatetradecane-1,3,43,45-tetracarboxylic acid (5.00 To a solution of 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (570 µg, 2.3 µmol) in DMF (500 µl) was added N,N-diisopropylethylamine (0.71 µl, 4.1 µmol; CAS-RN: 7087-68-5), and the reaction was stirred at room temperature for 3 hours. Water/0.1% TFA was added, and the mixture was filtered, and the filtrate was purified by preparative HPLC to give 3 mg of the product.

LC-MS (Method 4): R _t = 5.78 min; MS (ESIpos): m/z = 1254 [M+2Na] ²⁺ Intermediate 180

(3R,10S,13S,16S,19S,22R,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl} -22-[3-(2-bromoacetamido)propionamido]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyloxy-4,8,11,14,17,20,26,29,32,35,38,42-dodecanopentatetradecane-1,3,43,45-tetracarboxylic acid

(3R,10S,13S,16S,19S,22R,27S,30S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-22-[ [3-(2-Bromoacetamido)propionamido]-16,17,19,27,29,30-hexamethyl-5,9,12,15,18,21,25,28,31,34,37,41-dodecyl-4,8,11,14,17,20,26,29,32,35,38,42-dodecaazapentatetradecan-1,3,43,45-tetracarboxylic acid tetra-tributyl ester (12.2 mg, 4.89 µmol) was dissolved in 2,2,2-trifluoroethanol (4 ml). Zinc chloride (total 53.3 mg, 391 µmol) was added portionwise and the reaction was stirred at 50 °C for 24 hours. Afterwards, 4 mL of acetonitrile/water, 50 µL of TFA and ethylenediamine-N,N,N',N'-tetraacetic acid (121 mg, 416 µmol; CAS-RN: [60-00-4]) were added and the mixture was stirred for several minutes. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC to give 7 mg (63%) of the product. LC-MS (Method 2): R _t = 1.95 min; MS (ESI-): m/z = 1134 [M-2H] ^2- Intermediate 181

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-38-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol -1-yl)hexanoyl]amino}-16,17,19,59,61,62-hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoylhexadecane-1,3,75,77-tetracarboxylic acid

(3R,10S,13S,16S,19S,38S,59S,62S,65S,68S,75R)-38-amino-13,65-bis(2-amino-2-oxoethyl)-10,68-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-16,17,19 ,59,61,62-hexamethyl-5,9,12,15,18,21,37,41,57,60,63,66,69,73-tetradecanoyl-24,27,30,33,45,48,51,54-octaoxa-4,8,11,14,17,20,36,42,58,61,64,67,70,74-tetradecanoyl-1,3,75,77-tetracarboxylic acid)(7.80 To a solution of 1% 4-(2-nitropropy]-1-yl)-2- ^nitropropy ] -1 _...

(3R,10S,13S,16S,19S,27S,39S,42S,45S,48S,55R)-13,45-bis(2-amino-2-oxoethyl)-10,48-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-27-({2-[2-(2,5-dioxo- 2,5-Dihydro-1H-pyrrol-1-yl)acetamido]ethyl}aminomethyl)-16,17,19,39,41,42-hexamethyl-5,9,12,15,18,21,25,33,37,40,43,46,49,53-tetradecanoyl-4,8,11,14,17,20,26,32,38,41,44,47,50,54-tetradecanoylheptadecane-1,3,55,57-tetracarboxylic acid

N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}-L-propylamino-N-methyl-L-propylamino-N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide (10.0 mg, 8.37 µmol) in DMF (2.0 To a solution of N-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]ethyl}-L-dihydrocarbamide (2.48 mg, 4.18 µmol) in DMF and N,N-diisopropylethylamine (1.8 µl, 10 µmol; CAS-RN: 7087-68-5) were added and the reaction was stirred at room temperature. After stirring for 22 hours, the reaction was treated in an ultrasonic bath for 2 hours, followed by stirring at room temperature for 12 hours and in an ultrasonic bath for 2 hours. The mixture was concentrated to dryness under reduced pressure and the residue was purified by preparative HPLC to give 2 mg of the product. LC-MS (Method 1): R _t = 10.2 min; MS (ESIpos): m/z = 1243 [M+2H] ²⁺ Intermediate 183

(3R,10S,13S,16S,35S,56S,59S,62S,69R)-10,62-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-35-{[6-(2,5-dihydro-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino }-13,59-dimethyl-5,9,12,15,18,34,38,54,57,60,63,67-dodecyloxy-16,56-di(propan-2-yl)-21,24,27,30,42,45,48,51-octaoxa-4,8,11,14,17,33,39,55,58,61,64,68-dodecanoic acid heptadecane-1,3,69,71-tetracarboxylic acid

(3R,10S,13S,16S,35S,56S,59S,62S,69R)-10,62-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-35-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-13, 59-Dimethyl-5,9,12,15,18,34,38,54,57,60,63,67-dodecyloxy-16,56-di(propan-2-yl)-21,24,27,30,42,45,48,51-octaoxa-4,8,11,14,17,33,39,55,58,61,64,68-dodecahydroheptadecane-1,3,69,71-tetracarboxylic acid tetra-tributyl ester (31.2 mg, 11.2 µmol) was dissolved in 2,2,2-trifluoroethanol (3 ml). Zinc chloride (9 mg, 67.1 µmol) was added and the reaction was stirred at 50°C. After stirring for 1 hour, zinc chloride (9 mg, 67.1 µmol) was added and the reaction was stirred for another hour at 50°C. To drive the reaction to completion, zinc chloride (9 mg, 67.1 µmol) was added and the reaction was stirred for 1 hour at 50°C. Ethylenediamine-N,N,N',N'-tetraacetic acid (19.6 mg, 67.1 µmol; CAS-RN: [60-00-4]) was added and the mixture was stirred for a few minutes before water/0.1% TFA was added. The mixture was filtered and the filtrate was purified by preparative HPLC to give 10 mg of product. LC-MS (Method 4): R _t = 5.96 min; MS (ESIpos): m/z = 1306 [M+2Na] ²⁺ Intermediate 184

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-{[6-(2,5-difluorophenyl)-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl} 5-(5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-{[6-(2,5-dioxo-2,5-dihydro- Tetra-butyl 1H-pyrrol-1-yl)hexanoyl]amino}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decaoxy-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decaazanonatriacontane-1,3,37,39-tetracarboxylate (30.6 mg, 13.3 µmol) was dissolved in 2,2,2-trifluoroethanol (3 ml). Zinc chloride (10.9 mg, 79.9 µmol) was added and the reaction was stirred at 50°C for 1 hour. Zinc chloride (10.9 mg, 79.9 µmol) was added and the reaction was stirred at 50 °C for 1 hour. This step was repeated until the reaction was complete. Ethylenediamine-N,N,N',N'-tetraacetic acid (23.4 mg, 79.9 µmol; CAS-RN: [60-00-4]) was added and the mixture was stirred for a few minutes before adding water/0.1% TFA. The mixture was filtered and the filtrate was purified by preparative HPLC to give 15 mg of product. LC-MS (Method 4): R _t = 6.19 min; ESIpos): m/z = 1058 [M+2Na] ²⁺ Intermediate 185

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-19-({21-[(2,5-difluoropyrrolidin-1-yl)oxy]-17,21 -13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid

(3R,10S,13S,16S,19S,24S,27S,30S,37R)-19-[(15-amino-4,7,10,13-tetrahydropentadecan-1-yl)amino]-10,30-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-difluorophenyl Methylpropyl}(hydroxyacetyl)amino]ethyl}-13,27-dimethyl-5,9,12,15,18,22,25,28,31,35-decanoyl-16,24-di(propan-2-yl)-4,8,11,14,17,23,26,29,32,36-decazanonatriacontane-1,3,37,39-tetracarboxylic acid (10.0 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (3.64 mg, 11.2 µmol) in DMF (1.0 ml) was added N,N-diisopropylethylamine (3.1 µl, 18 µmol; CAS-RN: 7087-68-5) and the reaction was stirred at room temperature for 1 hour. Water/0.1% TFA was added and the mixture was purified by preparative HPLC to give 63 mg of the product.

LC-MS (Method 5): R _t = 5.19 min; MS (ESIpos): m/z = 1169 [M+2H] ²⁺ Intermediate 186

(3R,10S,13S,21S,33S,36S,43R)-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-21-{[ 2-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanyl}amino)ethyl]aminomethyl}-5,9,12,15,19,27,31,34,37,41-decanoyl-4,8,11,14,20,26,32,35,38,42-decazapentatetradecan-1,3,43,45-tetracarboxylic acid

To (3R,10S,13S,21S,33S,36S,43R)-21-[(2-aminoethyl)aminomethyl]-13,33-bis(2-amino-2-oxoethyl)-10,36-bis{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]ethyl}-5,9,12,15,19,27,31,34,37,41-decanoyl-4,8,11,14,20,26,32,35,38,42-decazapentatetradecan-1,3,43,45-tetracarboxylic acid (11.0 mg, 5.40 To a solution of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (4.41 mg, 13.5 µmol) and N,N-diisopropylethylamine (3.8 µl, 22 µmol; CAS-RN: 7087-68-5) in DMF (5.0 ml) was added, and the reaction was stirred at room temperature for 6 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 2 mg of the product.

LC-MS (method 1): R _t = 1.04 min; MS (ESIneg): m/z = 1122 [M+2H] ^2- A2DC Synthesis Part II: General Procedure A : Cysteine coupling and subsequent ring opening of the thiosuccinimide ring

Typically, a solution of 0.029 mg TCEP in 0.05 ml PBS buffer (pH 7.2) is added to 5 mg of the respective antibody (c=10 mg/ml) dissolved in 0.5 ml PBS under argon. (The concentration of the antibody solution can also vary from the standard procedure and range from 1 mg/ml to 30 mg/ml). The mixture is stirred at RT for 30 min, followed by an excess of 2 to 20 equivalents, preferably 2 to 10 equivalents, typically 7 equivalents (0.000233 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.05 mL DMSO. After stirring for another 90 min at RT, the mixture was diluted to a volume of 2.5 ml with PBS buffer pre-adjusted to pH 8, then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 8) and eluted with PBS buffer (pH 8). The solution was stirred at RT under argon at pH 8 overnight. Thereafter it was concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2) to a volume of approximately 1 to 5 mL. General Procedure A* : Cysteine Coupling and Subsequent Ring Opening of the Thiosuccinimide Ring

Typically, a solution of 0.172 mg TCEP in 0.25 ml PBS buffer (pH 7.2) was added to 30 mg of the respective antibody (c=10 mg/ml) dissolved in 3 ml PBS under argon. (The concentration of the antibody solution can also vary from the standard procedure and range from 1 mg/ml to 30 mg/ml). The mixture was stirred at RT for 30 min, followed by the addition of an excess of 2 to 10 equivalents, typically 7 equivalents (0.0014 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.25 mL DMSO. After stirring for another 90 min at RT, the mixture was diluted to a volume of 5 to 10 mL with PBS buffer pre-adjusted to pH 8, then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 8), and eluted with PBS buffer at pH 8. The solution was further diluted to a volume of 7.5 mL with PBS buffer (pH 8) and stirred overnight at RT under argon at pH 8. The mixture was then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 7.2), and eluted with PBS buffer (pH 7.2). The solution was then concentrated by ultracentrifugation and diluted to a volume of approximately 1 to 20 mL with PBS buffer (pH 7.2). General Procedure B : Cysteine Coupling

Typically, a solution of 0.029 mg TCEP in 0.05 ml PBS buffer (pH 7.2) is added to 5 mg of the respective antibody (c=10 mg/ml) dissolved in 0.5 ml PBS under argon. (The concentration of the antibody solution can also vary from the standard procedure and range from 1 mg/ml to 30 mg/ml). The mixture is stirred at RT for 30 min, followed by an excess of 2 to 20 equivalents, preferably 2 to 10 equivalents, typically 7 equivalents (0.000233 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.05 mL DMSO. After stirring for another 90 min at RT, the mixture was diluted to a volume of 2.5 ml with PBS buffer (pH 7.2), then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 7.2) and eluted with PBS buffer (pH 7.2). Thereafter, it was concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2) to a volume of approximately 1 to 5 mL. General Procedure B* : Cysteine Coupling

Typically, a solution of 0.115 mg TCEP in 0.2 ml PBS buffer (pH 7.2) was added to 20 mg of the respective antibody (c=14.8 mg/mL) dissolved in 1.35 ml PBS under argon. (The concentration of the antibody solution can also vary from the standard procedure and range from 1 mg/ml to 30 mg/ml). The mixture was stirred at RT for 30 min, followed by the addition of an excess of 2 to 10 equivalents, typically 7 equivalents (0.000933 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.2 mL DMSO. After stirring for another 90 min at RT, the mixture was diluted to a volume of 2.5 ml with PBS buffer (pH 7.2), then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 7.2), and eluted with PBS buffer (pH 7.2). Thereafter, it was concentrated by ultracentrifugation, diluted again with PBS buffer (pH 7.2) to a volume of approximately 14 mL, and concentrated again by ultracentrifugation to a final volume of approximately 1 to 20 mL. General Procedure C : Lysine Coupling

To a solution of 5 mg of the respective antibody, typically in 0.5 mL PBS buffer (pH 7.2) (c = 10 mg/mL) (the concentration of the antibody solution can also vary from the standard procedure and range between 1 mg/ml and 30 mg/ml) under argon atmosphere, an excess of 2 to 10 equivalents, preferably 2 to 5 equivalents, typically 5 equivalents (0.00165 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.5 mL DMSO is added. After stirring at RT for 30 to 60 min, the same amount of the ADC prodriver molecule dissolved in 0.05 mL DMSO is added and stirring at RT is continued for another 30 to 60 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of about 0.300 mL by ultracentrifugation and diluted again to a volume of about 1 to 5 mL with PBS buffer (pH 7.2). General Procedure C* : Lysine Coupling

To a solution of 5 mg of the respective antibody, typically in 0.5 mL PBS buffer (pH 7.2) (c = 10 mg/mL) (the concentration of the antibody solution can also vary from the standard procedure and range between 1 mg/ml and 30 mg/ml) under argon atmosphere, an excess of 2 to 10 equivalents, preferably 2 to 5 equivalents, typically 5 equivalents (0.00165 mmol) of the respective intermediate (ADC prodriver molecule) dissolved in 0.5 mL DMSO is added. After stirring at RT for 30 to 60 min, the same amount of the ADC prodriver molecule dissolved in 0.05 mL DMSO is added and stirring at RT is continued for another 30 to 60 min. Subsequently, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pH 7.2), applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer. The obtained solution was then concentrated to a volume of about 0.300 mL by ultracentrifugation and further diluted to a volume of 1 to 20 mL with PBS buffer (pH 7.2). Example II-1

As indicated in the table, ADCs were synthesized by coupling intermediate 158 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 158 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 1e-1015 HER-2 158 1015 A 1.45 2.6 99.2 1g-981 EGFR 158 981 A 1.63 2.4 1k-7007 TWEAKR 158 7007 A 1.72 3.2 1m-170 Mesothelin 158 170 A 1.36 3.1 96.3 1x-9574 CXCR5 158 9574 A 0.75 2.9 Example II-2

As indicated in the table, ADCs were synthesized by coupling intermediate 159 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 159 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 2e-1015 HER-2 159 1015 A 1.46 2.5 98.7 2g-981 EGFR 159 981 A 1.70 2.3 99.1 2k-7007 TWEAKR 159 7007 A 1.75 2.0 2m-170 Mesothelin 159 170 A 1.15 2.9 97.1 2x-9574 CXCR5 159 9574 A 1.08 2.3 2x-9574 CXCR5 159 9574 A* 1.59 2.5 Example II-3

As indicated in the table, ADCs were synthesized by coupling intermediate 160 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 160 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 3c-9476 CD123 160 9476 A 1.57 3.2 99.1 3e-1015 HER-2 160 1015 A 1.51 3.7 100 3g-981 EGFR 160 981 A 1.63 3.2 98.9 3k-7007 TWEAKR 160 7007 A 1.66 3.1 100 3x-9574 CXCR5 160 9574 A 0.59 3.7 100 Example II-4

As indicated in the table, ADCs were synthesized by coupling intermediate 161 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 161 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 4c-9476 CD123 161 9476 A 1.64 3.5 98.8 4e-1015 HER-2 161 1015 A 1.58 3.9 100 4g-981 EGFR 161 981 A 1.60 3.9 100 4k-7007 TWEAKR 161 7007 A 1.58 3.5 99 4x-9574 CXCR5 161 9574 A 0.27 4.0 97.1 Example II-5

As indicated in the table, ADCs were synthesized by coupling intermediate 162 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 162 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 5c-9476 CD123 162 9476 A 1.57 3.4 99 5e-1015 HER-2 162 1015 A 1.54 3.5 100 5g-981 EGFR 162 981 A 1.63 3.4 98.8 5k-7007 TWEAKR 162 7007 A 1.59 3.2 100 5x-9574 CXCR5 162 9574 A 0.48 3.7 100 Example II-6

As indicated in the table, ADCs were synthesized by coupling intermediate 163 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 163 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 6e-1015 HER-2 163 1015 A 1.65 2.5 100 6g-981 EGFR 163 981 A 1.72 2.2 99.1 6k-7007 TWEAKR 163 7007 A 1.68 2.0 100 6m-170 Mesothelin 163 170 A 0.77 3.5 97.7 6m-170* Mesothelin 163 170 A* 4.09 4.3 92.3 Example II-6a Metabolite 1

The synthesis of this isomeric non-permeable metabolite 1 released from A2DC in Example 6 has been described in WO2018/114804. Example II-6b Metabolite 2

The synthesis of this permeable metabolite 2 released from A2DC in Example 6 was synthesized according to the following procedure.

Step 1 : To a solution of (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid (40.0 mg, 60.8 µmol) in DMF (7 ml) were added 3-amino-N-methylpropionamide*HCl (10.1 mg, 73.0 µmol), HATU (27.7 mg, 73.0 µmol; CAS-RN: 148893-10-1) and N,N-diisopropylethylamine (32 µl, 182 µmol; CAS-RN: 7087-68-5), and the reaction mixture was stirred at room temperature. h. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC to give 37.5 mg (83% yield, 100% purity) of 2-(trimethylsilyl)ethyl {(2S)-1-[(3-methylamino-3-oxopropyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-oxobutyl-2-yl}carbamate.

LC-MS (Method 6): Rt = 1.32min; MS (ESIpos): m/z = 741 (M+H) ⁺

Step 2 : 2-(Trimethylsilyl)ethyl {(2S)-1-[(3-methylamino-3-oxopropyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-oxobutyl-2-yl}carbamate (37.5 mg, 50.5 µmol) was dissolved in 2,2,2-trifluoroethanol (5 ml). Zinc chloride (41.3 mg, 303 µmol) was added and the reaction was stirred at 50 °C for 2.5 hours. Ethylenediamine-N,N,N',N'-tetraacetic acid (88.6 mg, 303 µmol; CAS-RN: [60-00-4]) was added, followed by acetonitrile and water, and the mixture was stirred for several minutes. The mixture was filtered and the filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 26 mg (70.5% yield, 97.5% purity) of (2S)-2-amino-N-(3-methylamino-3-oxopropyl)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]butyramide-trifluoroacetic acid.

LC-MS (Method 6): Rt = 0.77 min; MS (ESIpos): m/z = 598 (M+H) ⁺ Example II-7

As indicated in the table, ADCs were synthesized by coupling intermediate 164 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 164 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 7c-9476 CD123 164 9476 A 2.19 2.9 98.6 7e-1015 HER-2 164 1015 A 2.10 3.2 97.6 7g-981 EGFR 164 981 A 1.98 3.3 94.3 7k-7007 TWEAKR 164 7007 A 2.16 2.8 98.2 7x-9574 CXCR5 164 9574 A 0.8 2.2 100 Example II-8

As indicated in the table, ADCs were synthesized by coupling intermediate 165 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 165 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 8e-1015 HER-2 165 1015 C 1.23 1.3 98.5 8g-981 EGFR 165 981 C 1.22 1.2 98.4 8k-7007 TWEAKR 165 7007 C 1.70 2.4 97.7 8c-9476 CD123 165 9476 C 1.43 3.3 97.7 Example II-9

As indicated in the table, ADCs were synthesized by coupling intermediate 166 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 166 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 9e-1015 HER-2 166 1015 B 1.87 2.8 98.2 9g-981 EGFR 166 981 B 2.13 2.7 96.9 9k-7007 TWEAKR 166 7007 B 2.12 2.9 100 9c-9476 CD123 166 9476 B 2.33 2.8 100 Example II-10

As indicated in the table, ADCs were synthesized by coupling intermediate 167 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 167 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 10e-1015 HER-2 167 1015 C 0.81 4.0 94.8 10g-981 EGFR 167 981 C 2.07 3.7 100 10k-7007 TWEAKR 167 7007 C 2.37 4.2 100 Example II-11

As indicated in the table, ADCs were synthesized by coupling intermediate 169 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 169 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 11e-1015 HER-2 168 1015 B 2.2 3.2 100 11m-170 Mesothelin 168 170 B 1.19 3.8 97.9 11m-170* Mesothelin 168 170 B* 6.58 4.7 96.6 11k-7007 TWEAKR 168 7007 B 1.43 3.0 100 11k-2658 TWEAKR 168 2658 B 2.32 4.2 100 11k-2658* TWEAKR 168 2658 B* 7.8 3.6 98.8 11k-2090* TWEAKR 168 2090 B* 7.97 1.3 85.8 Example II-12

As indicated in the table, ADCs were synthesized by coupling intermediate 169 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 169 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 12e-1015 HER-2 169 1015 A 1.73 2.7 95.3 12x-9574 CXCR5 169 9574 A 0.52 3.3 95.5 12k-7007 TWEAKR 169 7007 A 1.93 1.5 100 Example II-13

As indicated in the table, ADCs were synthesized by coupling intermediate 170 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 170 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 13e-1015 HER-2 170 1015 C 0.7 2.4 97.2 13g-981 EGFR 170 981 C 1.3 2.3 97.6 13k-7007 TWEAKR 170 7007 C 1.86 2.8 93.2 13c-9476 CD123 170 9476 C 1.31 3.3 96.4 Example II-14

As indicated in the table, ADCs were synthesized by coupling intermediate 171 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 171 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 14e-1015 HER-2 171 1015 C 0.67 3.8 100 14g-981 EGFR 171 981 C 2.4 3.1 91.0 14k-7007 TWEAR 171 7007 C 2.37 2.7 94.5 14c-9476 CD123 171 9476 C 2.5 4.5 94.8 14x-9574 CXCR5 171 9574 C 0.41 2.9 100 Example II-15

As indicated in the table, ADCs were synthesized by coupling intermediate 172 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 172 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 15m-170 Mesothelin 172 170 B 1.42 3.6 95.8 15e-1015 HER-2 172 1015 B 1.92 3.1 100 15g-981 EGFR 172 981 B 2.25 3.2 100 15k-7007 TWEAR 172 7007 B 2.22 3.3 100 15x-9574 CXCR5 172 9574 B 0.93 1.6 100 15x-9574 CXCR5 172 9574 B* 3.86 1.7 96.7 15c-9476 CD123 172 9476 B 2.16 3.0 100 Example II-16

As indicated in the table, ADCs were synthesized by coupling intermediate 173 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 173 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 16e-1015 HER-2 173 1015 B 2.19 3.1 100 16g-981 EGFR 173 981 B 2.21 3.0 100 16k-7007 TWEAKR 173 7007 B 2.24 2.6 100 16x-9574 CXCR5 173 9574 B 1.04 2.8 100 16c-9476 CD123 173 9476 B 2.25 2.8 100 Example II-17

As indicated in the table, ADCs were synthesized by coupling intermediate 174 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 174 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 17e-1015 HER-2 174 1015 C 1.77 3.4 95.1 17g-981 EGFR 174 981 C 2.99 3.9 100 17k-7007 TWEAKR 174 7007 C 2.86 3.3 93.8 17x-9574 CXCR5 174 9574 C 0.5 2.6 83.5 17c-9476 CD123 174 9476 C 2.89 2.8 98.2 Example II-18

As indicated in the table, ADCs were synthesized by coupling intermediate 175 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 175 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/m] DAR Single % 18e-1015 HER-2 175 1015 C 1.22 4.4 99.5 18k-7007 TWEAKR 175 7007 C 1.47 3.1 100 18x-9574 CXCR5 175 9574 C 1.24 3.2 96.7 Example II-19

As indicated in the table, ADCs were synthesized by coupling intermediate 176 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 176 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 19e-1015 HER-2 176 1015 A 1.99 2.9 100 19k-7007 TWEAKR 176 7007 A 1.63 2.6 100 19x-9574 CXCR5 176 9574 A 0.38 2.5 100 Example II-20

As indicated in the table, ADCs were synthesized by coupling intermediate 178 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 178 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 20e-1015 HER-2 177 1015 C 1.96 2.5 100 20g-981 EGFR 177 981 C 2.06 2.1 100 20k-7007 TWEAKR 177 7007 C 2.05 2.0 99.1 20x-9574 CXCR5 177 9574 C 1.1 2.8 100 Example II-21

As indicated in the table, ADCs were synthesized by coupling intermediate 178 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 178 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 21e-1015 HER-2 178 1015 B 1.97 3.5 96.1 21g-981 EGFR 178 981 B 2.2 3.6 99 21k-7007 TWEAKR 178 7007 B 2.17 3.5 98.3 21c-9476 CD123 178 9476 B 2.0 3.4 96.5 Example II-22

As indicated in the table, ADCs were synthesized by coupling intermediate 179 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 179 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 22e-1015 HER-2 179 1015 A 1.66 3.2 96.9 22g-981 EGFR 179 981 A 1.71 3.0 96.3 22k-7007 TWEAKR 179 7007 A 1.96 3.1 96.4 22c-9476 CD123 179 9476 A 1.68 3.1 96.9 Example II-23

As indicated in the table, ADCs were synthesized by coupling intermediate 180 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 180 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 23k-7007 TWEAKR 180 7007 B 1.71 2.4 87.7 23x-9574 CXCR5 180 9574 B 0.71 2.0 100 Example II-24

As indicated in the table, ADCs were synthesized by coupling intermediate 181 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 181 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 24e-1015 HER-2 181 1015 B 2.06 3.5 100 24g-981 EGFR 181 981 B 2.17 3.5 100 24k-7007 TWEAKR 181 7007 B 2.03 3.3 100 24c-9476 CD123 181 9476 B 2.12 3.3 100 Example II-25

As indicated in the table, ADCs were synthesized by coupling intermediate 182 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 182 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 25e-1015 HER-2 182 1015 A 1.2 4.5 100 25k-7007 TWEAKR 182 7007 A 1.57 3.0 100 25x-9574 CXCR5 182 9574 A 0.46 3.0 100 Example II-26

As indicated in the table, ADCs were synthesized by coupling intermediate 183 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 183 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 26m-170 Mesothelin 183 170 B 1.46 4.0 98.4 26k-2658 TWEAKR 183 2658 B 2.45 3.9 100 26x-9574 CXCR5 183 9574 B 1.29 3.1 94.6 26k-7007 TWEAKR 183 7007 B* 15.12 4.0 96.4 26c-9476 CD123 183 9476 B* 14.19 3.4 89.2 Example II-27

As indicated in the table, ADCs were synthesized by coupling intermediate 184 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 184 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 27k-2658 TWEAKR 184 2658 B 4.49 4.0 100 27x-9574 CXCR5 184 9574 B 0.34 1.6 96.7 27c-6013 CD123 184 6013 B 2.34 3.9 100 Example II-28

As indicated in the table, ADCs were synthesized by coupling intermediate 185 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 185 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 28g-981 EGFR 185 981 C 2.01 2.7 98.9 28e-1015 HER-2 185 1015 C 1.81 2.7 100 28x-9574 CXCR5 185 9574 C 1.01 3.5 100 28k-7007 TWEAKR 185 7007 C 2.07 2.2 100 28c-9476 CD123 185 9476 C 2.08 3.6 98.4 Example II-29

As indicated in the table, ADCs were synthesized by coupling intermediate 186 to different antibodies using one of the general procedures described above. The characteristics of the ADC batches obtained by coupling intermediate 186 to different antibodies are shown in the following table: Example II- Target Intermediate mAb TPP- program C [mg/mL] DAR Single % 29g-981 EGFR 186 981 C 2.64 3.9 97.2 29e-1015 HER-2 186 1015 C 2.08 3.8 97.1 29x-9574 CXCR5 186 9574 C 1.14 3.6 100 29k-7007 TWEAKR 186 7007 C 2.70 3.9 97.7 29c-9476 CD123 186 9476 C 2.47 4.5 95.9 Biological Characterization CTG Analysis

Cells were grown according to standard methods using the growth medium listed under CI. The assay was performed as follows: cells were detached with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelleted, resuspended in medium, counted and seeded in 96-well culture plates with white bottom (Costar #3610) (75 pl/well, the number of cells per well was as follows: NCI-H292: 2500 cells/well, BxPC3 2500 cells/well, LoVo 3000 cells/well) and incubated in an incubator at 37°C and 5% carbon dioxide. After 24 h, 25 pi medium (fourfold concentration) containing antibody-drug conjugate was added to the cells, so that the final concentration of antibody-drug conjugate on the cells reached 3×10 ^-7 M to 3×10 ^-11 M (triplicate). The cells were then cultured in an incubator at 37°C and 5% carbon dioxide. On a parallel culture plate, at the beginning of drug treatment (day 0), cell activity was measured using the Cell Titer Glow (CTG) luminescent cell viability assay (Promega #G7573 and #G7571). For this purpose, 100 pi of substrate was added to each batch of cells, followed by covering the culture plates with aluminum foil, shaking on a plate shaker at 180 rpm for 2 minutes, standing on the bench for 8 minutes, and then measured using a luminometer (Victor X2, Perkin Elmer). The substrate detects the ATP content in living cells, generating a luminescent signal whose intensity is proportional to cell viability. After incubation with the antibody-drug conjugate for 72 h, the viability of these cells was then determined using the Cell Titer Glow luminescent cell viability assay described above. Based on the measured data, the IC50 for growth inhibition compared to day 0 was calculated using the DRC (dose response curve) analysis spreadsheet and 4-parameter fitting. The DRC analysis spreadsheet is a Biobook spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).

The activity data reported are for the examples described in the experimental section of the present invention, where the drug/mAB ratios are indicated. The values may deviate due to different drug/mAB ratios. The IC50 values are the average of several independent experiments or individual values. The effect of the antibody drug conjugates is selective for the respective isotype control comprising the respective linker and toxin group.

The following Tables 9a to 9f list the IC50 values of representative examples from this analysis: Table 9a : TWEAKR-A2DC Instance Number (II-) EO-PROLIF-NCI-H292 ADC CTG 72H IC50 - [mol/l] (average) EO-Prolif-BXPC3 ADC CTG 72h IC50 - [mol/l] (average) EO-PROLIF-LoVo ADC CTG 72H IC50 - [mol/l] (average) 1k-7007 6.35 E-11 6.14 E-11 ＜ 3.00 E-11 2k-7007 9.79 E-11 1.02 E-10 ＜ 3.00 E-11 3k-7007 6.18 E-11 7.67 E-11 3.00 E-11 ^* 4k-7007 5.78 E-11 1.73 E-10 ＜ 3.00 E-11 5k-7007 4.34 E-11 8.46 E-11 ＜ 3.00 E-11 6k-7007 1.17 E-10 1.39 E-10 ＜ 3.00 E-11 7k-7007 1.49 E-10 1.98 E-10 ＜ 3.00 E-11 8k-7007 9.82 E-11 1.78 E-10 ＜ 3.00 E-11 9k-7007 1.18 E-10 1.82 E-10 ＜ 3.00 E-11 10k-7007 4.25E-11 ^* 7.12 E-11 ^* 3.00 E-11 ^* 11k-2090* 5.28 E-10 9.28 E-10 2.13 E-10 11k-2658 2.76E-10 3.11 E-10 4.64 E-11 11k-7007 3.31 E-11 6.10 E-11 ＜ 3.00 E-11 12k-7007 9.31 E-11 1.78 E-10 ＜ 3.00 E-11 13k-7007 9.03 E-11 2.23 E-10 ＜ 3.00 E-11 14k-7007 6.52 E-11 1.30 E-10 ＜ 3.00 E-11 15k-7007 6.72 E-11 1.33 E-10 ＜ 3.00 E-11 16k-7007 8.48 E-11 1.27 E-10 ＜ 3.00 E-11 17k-7007 4.10 E-11 7.50 E-11 ＜ 3.00 E-11 19k-7007 5.87 E-11 9.17 E-11 ＜ 3.00 E-11 20k-7007 8.02 E-11 1.77 E-10 ＜ 3.00 E-11 21k-7007 6.85 E-11 6.83 E-11 ＜ 3.00 E-11 22k-7007 7.71 E-11 7.89 E-11 ＜ 3.00 E-11 23k-7007 7.91 E-11 7.02 E-11 ＜ 3.00 E-11 24k-7007 ＜ 3.00 E-11 ＜ 3.00 E-11 ＜ 3.00 E-11 25k-7007 ＜ 3.00 E-11 ＜ 3.00 E-11 ＜ 3.00 E-11 26k-2658 1.23 E-10 1.60 E-10 ＜ 3.00 E-11 26k-7007* 5.87 E-11 1.21 E-10 ＜ 3.00 E-11 27k-2658 3.01 E-10 4.79E-10 8.01 E-11 28k-7007 7.66 E-11 2.33 E-10 ＜ 3.00 E-11 29k-7007 7.59 E-11 8.75 E-11 3.00 E-11 ^* Table 9b : Mesothelin -A2DC Instance Number (II-) Cytotoxicity assay IC50 [mol/l] HT29 meso (average value) Cytotoxicity assay IC50 [mol/l] H322 (average value) 1m-170 2.13 E-9 2m-170 2.20 E-9 6m-170 5.71 E-10 3.72 E-9 6m-170* 2.84 E-10 11m-170 1.27 E-10 5.73 E-9 11m-170* 1.05 E-8 15m-170 1.01 E-10 3.48 E-9 26m-170 4.94 E-10 2.36 E-9 Table 9c : EGFR-A2DC Instance Number (II-) Cytotoxicity assay IC50 [mol/l] H292 (geometric mean) Cytotoxicity assay IC50 [mol/l] SKHep-1 (average value) 4g-981 7.24 E-12 4.12 E-12 7g-981 5.87 E-13 4.40 E-12 8g-981 1.00 E-12 1.87 E-12 9g-981 1.58 E-12 1.92 E-11 10g-981 5.74 E-13 2.35 E-12 13g-981 1.84 E-12 2.88 E-12 14g-981 1.00 E-11 1.00 E-11 15g-981 1.00 E-11 7.16 E-12 16g-981 1.00 E-13 2.07 E-12 17g-981 1.86 E-13 5.27 E-13 20g-981 1.00 E-11 3.58 E-12 21g-981 1.14 E-13 1.00 E-12 22g-981 1.32 E-13 1.03 E-12 24g-981 2.04 E-12 2.16 E-11 29g-981 2.59 E-13 1.00 E-12 Table 9d : HER2-ADC Instance Number (II-) Cytotoxicity assay IC50 [mol/l] KPL4 (average value) 1e-1015 1.93 E-11 2e-1015 1.56 E-11 3e-1015 2.37 E-11 4e-1015 1.64 E-10 5e-1015 9.62 E-11 6e-1015 8.36 E-11 7e-1015 2.08 E-10 8e-1015 2.34 E-10 9e-1015 1.55 E-10 10e-1015 1.23 E-10 11e-1015 9.53 E-11 12e-1015 1.23 E-10 13e-1015 4.23 E-10 14e-1015 9.63 E-11 15e-1015 5.22 E-11 16e-1015 7.15 E-11 17e-1015 1.02 E-10 19e-1015 3.09 E-11 20e-1015 9.45 E-11 21e-1015 8.23 E-11 22e-1015 5.68 E-11 24e-1015 6.81 E-12 25e-1015 2.83 E-11 28e-1015 1.74 E-10 29e-1015 3.42 E-11 Table 9e : CXCR5-A2DC Instance Number (II-) EO-PROLIF-Rec-1_ADC_CTG_72h IC50 - [mol/l] (mean) 1x-9574 3.17 E-12 2x-9574* 6.29 E-12 2x-9574 3.04 E-12 3x-9574 ＜ 3.00 E-12 4x-9574 ＜ 3.00 E-12 5x-9574* ＜ 3.00 E-12 5x-9574 ＜ 3.00 E-12 6x-9574* 3.41 E-12 ^* 7x-9574 ＜ 3.00 E-11 12x-9574 8.09 E-12 14x-9574 ＜ 3.00 E-11 15x-9574* 9.83E-12 ^* 15x-9574 ＜ 3.00 E-11 16x-9574 ＜ 3.00 E-11 17x-9574 8.02 E-11 19x-9574 5.51 E-10 20x-9574 ＜ 3.00 E-11 23x-9574 ＜ 3.00 E-11 25x-9574 ＜ 3.00 E-11 26x-9574 2.10E-11 ^* 27x-9574 3.27 E-11 ^* 28x-9574 3.91 E-11 29x-9574 7.24 E-12 Table 9f : CD123-A2DC Instance Number (II-) Cytotoxicity assay IC50 [mol/l] MOLM-13 (average value) Cytotoxicity assay IC50 [mol/l] MV-4-11 (average value) 3c-9476 4c-9476 4.18 E-9 8.57 E-8 5c-9476 8.66 E-11 2.48 E-11 7c-9476 2.07 E-8 7.93 E-9 8c-9476 5.36 E-9 2.69 E-10 9c-9476 1.39 E-8 2.36 E-9 13c-9476 9.29 E-10 9.39 E-11 14c-9476 1.50 E-9 3.07 E-11 15c-9476 2.70 E-10 7.67 E-11 16c-9476 5.00 E-7 5.00 E-7 21c-9476 1.01 E-9 1.83 E-10 22c-9476 5.24 E-9 1.26 E-9 24c-9476 1.21 E-9 3.13 E-10 26c-9476* 3.92 E-11 5.36 E-11 27c-6013 1.50 E-11 2.14 E-11 28c-9476 6.50 E-10 3.47 E-10 29c-9476 5.00 E-7 5.00 E-7 MTT assay

Cells were grown according to standard methods using the growth medium listed under CI. The assay was performed as follows: cells were detached with a solution of Accutase in PBS (from Biochrom AG #L2143), pelletized, resuspended in medium, counted and seeded in 96-well plates with white bottom (from Costar #3610) (NCI H292: 2500 cells/well; SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well; total volume 100 pi). Cells were then grown in an incubator at 37°C and 5% CO2. After 48 h, the medium was replaced. Then 10 μl of medium containing the antibody-drug conjugate at a concentration of 10 ^-5 M to 10 ^-13 M was pipetted onto the cells (repeated three times), and the analytes were then incubated in an incubator at 37 ° C and 5% carbon dioxide. After 96 h, cell proliferation was detected using the MTT assay (ATCC, Manassas, Va., USA, catalog number 30-1010K). For this purpose, the MTT reagent was incubated with the cells for 4 hours, after which the cells were lysed overnight by adding a detergent. The dye formed was detected at 570 nm (Infinite M1000 pro, Tecan). Using DRC (dose response curve), the IC50 of growth inhibition was calculated using the measured data. The proliferation of cells not treated with the test substance but otherwise treated identically was defined as 100% of the figure. Table 10 : Designated Example 11e compared to the respective isotype control A2DC :

Highly potent trastuzumab-A2DC; has exceptionally high potency and selectivity relative to the isotype control A2DC. Examples DAR KPL 4 EC50 [nM] SKOV 3 EC50 [nM] NCI N87 EC50 [nM] SK-BR3 EC50 [nM] II-11e-1015 3.2 0.1 0.006 0.04 0.003 Isotype control A2DC 3.7 500 500 500 500 Table 11 : Designated Example 11m-170 : A2DC retains bystander toxicity of the permeable ADC component : A2DC is potent against mesothelin - transfected HT29 cells, HT29 wild-type, and co-cultures compared to isotype control A2DC and the individual ADC components in A2DC Examples DAR HT29/Meso ⁺ EC50 [nM] HT29/Meso ^- EC50 [nM] HT29 mixture Meso ⁺ /Meso ^- EC50 [nM] II-11m-170 3.8 0.8 >300 5 Reference ADC with permeable metabolites 6.1 1 226 2 Reference ADC with Impermeable Metabolites 7.3 32 >300 >300 Table 12 : Designated Examples 15m-170 : A2DC is more potent than individual ADCs against mesothelin-transfected HT29 cells and also depends on target expression. Examples DAR HT29/Meso ⁺ EC50 [nM] HT29/Meso ^- EC50 [nM] HT29 mixture Meso ⁺ /Meso ^- EC50 [nM] II-15m-170 3.6 0.1 >300 58 Reference ADC with Impermeable Metabolites 4.9 5.4 >300 >300 Internalization Analysis

Internalization is a key process that enables the antibody drug conjugate (ADC) to be loaded specifically and effectively into cancer cells expressing the antigen. This process is monitored by fluorescent labeling of specific antibodies and isotype control antibodies. First, the fluorescent dye is conjugated to the lysine of the antibody. Conjugation is performed using a two-fold molar excess of CypFier 5E mono-NFiS ester (lot 357392, GE Firecare) at pH 8.3. After coupling, the reaction mixture was purified by gel chromatography (Zeba spin desalination column, 40K, Thermo Scientific, No. 87768; elution buffer: DULBECCO'S PBS, Sigma-Aldrich, No. D8537) to eliminate excess dye and adjust the pH. The protein solution was concentrated using a VIVASPIN 500 column (Sartorius stedim biotec). The dye loading of the antibody was determined by spectrophotometric analysis (from NanoDrop) and subsequent calculation (D/P = A _dye ε _protein : (A ₂₈₀ -0.16A _dye )ε _dye ).

The dye loading of the antibodies and isotype controls tested here was of similar order of magnitude. In the cell binding assay, it was demonstrated that the conjugation did not cause a change in antibody affinity.

Labeled antibodies were used for internalization analysis. Before starting treatment, cells (2×10 ⁴ /well) were seeded in 96-well MTP (coarse, black, clear bottom, No. 4308776, from Applied Biosystems) in 100 pi medium. After incubation for 18 h at 37°C/5% CO2, the medium was replaced and labeled antibodies were added at different concentrations (10, 5, 2.5, 1, 0.1 pg/ml). The same treatment scheme was applied to the labeled isotype control (negative control). The selected incubation times were 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6 h and 24 h. Fluorescence measurements were performed using InCelAnalyzer 1000 (from GE Healthcare). Kinetic evaluation was then performed by measuring the parameters particle count/cell and total particle density/cell.

After binding to the receptor, the internalization capacity of the antibodies was tested. For this purpose , cells with different receptor expression levels were selected. Target-mediated specific internalization of the antibodies was observed, while isotype controls showed no internalization. Quantification of metabolites formed by A2DC

To quantify A2DC metabolites released from A2DC, cancer cells were incubated with A2DC from Example 6 for 72 h. Cell lysates and corresponding supernatants were collected at each time point, and active payload metabolites were quantified by LC-MS. For calibration, 0.6 to 1000 µg/L active metabolite 8 was added to cell homogenates (for cell lysates) or cell culture medium (for supernatants). Samples were analyzed using HPLC coupled to a triple quadrupole mass spectrometer API 4500 (AB Sciex).

In order to demonstrate the formation of individual metabolites, A2DC Example II-6 was studied in this analysis, and the metabolites described in Examples II 6a and 6b as reference were detected. Metabolites with low cell permeability were detected in the cell fraction (Example II-6a), and permeable metabolites were also detected in the supernatant (Example II-6b). The results are shown in Figure 5. Growth inhibition / regression of experimental tumors in mice

Murine tumor cells expressing the antigen of the antibody drug conjugate are inoculated subcutaneously into the flank of immunocompetent mice (e.g., Balb/c or C57Bl/6 mice). One million to ten million cells are isolated from the cell culture, centrifuged and resuspended in medium or medium/matrix gel. The cell suspension is injected subcutaneously into the mouse. Within a few days, tumors grow. Treatment is initiated after tumors are established and are approximately 40 ^mm2 in size. To test the effect on larger tumors, treatment may be initiated only when tumors are 50-100 ^mm2 in size. A2DC treatment is performed intraperitoneally or via the intravenous (iv) route to the tail vein of the mouse. A2DC was administered at a volume of 5 ml/kg.

The treatment regimen depends on the pharmacokinetic of the antibody. For the conjugates according to the invention, as a standard, treatment is carried out once or twice a week for 2 or 3 weeks. For highly effective compounds, a regimen with a single treatment can be adopted. However, the treatment can also be continued, or a second cycle with three treatment days can be continued later. As a standard, 8 animals are used per treatment group. In addition to the group to which the active substance is administered, one group is treated only with buffer according to the same regimen as a control group. During the experiment, the two-dimensional area of the tumor (length/width) is measured regularly using a caliper. The tumor area is measured as length×width. The ratio of the mean tumor area of the treatment group to the mean tumor area of the control group was expressed as T/C _area .

Syngeneic (mouse) tumor models CT26 and MC38 were studied, both of which express TWEAK receptors as confirmed by immunohistochemistry. A2DC Example II-11k-2658* was injected intraperitoneally into CT26 tumor-bearing mice (Balb/c, female) at two different doses (2.5 and 5 mg/kg) in a single treatment, and tumor growth was observed to last for about 3 weeks. Therefore, a stronger dose-dependent tumor growth inhibition of 11k-2658 could be demonstrated. A similar stronger efficacy of Example II-11k-2658* was found in MC38 tumor-bearing mice treated three times with 5 mg/kg. (Figure X1)

In both studies, the A2DC compound was well tolerated and did not affect the body weight or behavior of the rodents. There were no suspicious findings at gross necropsy.

A summary of the T/C _area ratios from these studies is listed below ( Table 13 ). Table 13 : Summary of T/C ratios from different tumor models A2DC Example (II-) Tumor Model Dosage T/C _area ( days ) 11k-2658* CT26 2.5 mg/kg, SD 0.30 (d17) 11k-2658* CT26 5 mg/kg, SD 0.15 (d17) 11k-2658* MC38 5 mg/kg, q3dx3 0.26 (d18)

Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The following claims are intended to define the scope of the present invention and therefore cover methods and structures within the scope of this claim and its equivalents.

FIG. 1 shows the efficacy of A2DC on mesothelin-transfected HT29 cells and HT29 wild-type cells compared to the isotype control A2DC and the individual ADC components in A2DC (Example I-11m).

FIG. 2 shows bystander killing of A2DC that retains permeable ADC components: The potency of A2DC (Example 1-6m) against mesothelin-transfected HT29 cells, HT29 wild type, and co-cultures compared to the individual ADC components in A2DC.

FIG. 3 shows tumor growth in NCI-H292 xenografts after treatment with A2DC.

FIG. 4 shows tumor growth of CT26 (syngeneic model) after treatment with A2DC.

FIG. 5 shows the analysis of different metabolites in and outside cells after incubation of mesothelin-transfected HT29 cells with A2DC of Example II-6m.

FIG. 6 shows tumor growth of CT26 (syngeneic model) after treatment with A2DC.

TW202434289A_112144384_SEQL.xml

Claims (137)

An antibody-drug conjugate of formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof: (I) (II) (III) wherein: ^AK1 is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via the sulfur atom of cysteine, an antibody linked via the nitrogen atom of lysine, or an antibody linked via both the sulfur atom of cysteine and the nitrogen atom of lysine); ^AK2 and ^AK3 are each independently an antibody or an antigen-binding fragment thereof linked via the sulfur atom of cysteine or via the nitrogen atom of lysine; ^X1 and ^X2 are each independently -NH- or -S-; ^L1 , ^L2 , ^L3 , and ^L4 are each independently a stabilizing linker; ^RA is a first protease-cleavable spindle-dried protein inhibitor group; ^RB is a second protease-cleavable spindle-dried protein inhibitor group; wherein ^RA and ^RB are different groups; ^RC and R ^D is each independently a protease-cleavable spindle-dried protein inhibitor; P ⁵ is a protease-cleavable linker; KSP ⁵ is a first spindle-dried protein inhibitor; KSP ⁶ is a second spindle-dried protein inhibitor; and a, b, y and z are each independently an integer from 1 to 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein the antibody-drug conjugate has the structure of formula (I): (I) wherein: AK ¹ is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via the sulfur atom of cysteine, an antibody linked via the nitrogen atom of lysine, or an antibody linked via both the sulfur atom of cysteine and the nitrogen atom of lysine); X ¹ and X ² are each independently -NH- or -S-; L ¹ and L ² are each independently a stabilizing linker; ^RA is a first protease-cleavable spinning hammer-driving protein inhibitor group; ^RB is a second protease-cleavable spinning hammer-driving protein inhibitor group; wherein ^RA and ^RB are different groups; and a and b are each independently an integer from 1 to 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 1 or 2, wherein: ^RA is a first protease-cleavable spinning hammer protein inhibitor of formula (i): (i) KSPi ¹ -P ¹ - ^# ; wherein KSPi ¹ is a first spinning hammer protein inhibitor, P ¹ is a first protease-cleavable linker; and ^# is a bond between ^RA and L ¹ ; and ^RB is a second protease-cleavable spinning hammer protein inhibitor of formula (ii): (ii) ^## -P ² -KSPi ² ; wherein ^## is a bond between L ² and ^RB ; P ² is a second protease-cleavable linker; KSPi ² is a second spinning hammer protein inhibitor, and wherein ^RA and ^RB are different groups. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the antibody-drug conjugate has a structure of formula (I'): (I') wherein: AK ¹ is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via the sulfur atom of cysteine, an antibody linked via the nitrogen atom of lysine, or an antibody linked via both the sulfur atom of cysteine and the nitrogen atom of lysine); X ¹ and X ² are each independently -NH- or -S-; L ¹ and L ² are each independently: , , , , , , , , or , ^P1 is a first protease-cleavable linker; ^P2 is a second protease-cleavable linker; KSPi ¹ is: ; KSPi ² is: ; R ¹ and R ⁶ are each independently a halogen; R ² and R ⁷ are each independently a C _1-6 alkyl; m and n are each independently an integer from 1 to 5; and a and b are each independently an integer from 1 to 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein ^P1 and ^P2 are each independently a legumain-cleavable linker. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein ^P1 and ^P2 are each independently a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein: ^AK1 is an antibody or an antigen-binding fragment thereof (e.g., an antibody linked via a sulfur atom of cysteine, an antibody linked via a nitrogen atom of lysine, or an antibody linked via both a sulfur atom of cysteine and a nitrogen atom of lysine); ^X1 and ^X2 are each independently -NH- or -S-; ^L1 and ^L2 are each independently: , , , , , , , , or ; ^RA is: ; ^RB is: ; wherein: R ¹ and R ⁶ are each independently a halogen; R ² and R ⁷ are each independently a C _1-6 alkyl; R ³ and R ⁸ are each independently a C _1-6 alkyl or -C _1-6 alkylene-COOH; R ⁴ , R ⁵ , R ⁹ and R ¹⁰ are each independently hydrogen or C _1-6 alkyl; a and b are each independently an integer from 1 to 50; m and n are each independently an integer from 1 to 5; and p, q, r and s are each independently 0, 1, 2, 3 or 4. The antibody-drug conjugate of claim 1, wherein ^X1 and ^X2 are each -NH-. The antibody-drug conjugate of claim 1, wherein ^X1 and ^X2 are each -S-. The antibody-drug conjugate of claim 1, wherein X ¹ is -NH- and X ² is -S-. The antibody-drug conjugate of claim 1, wherein ^X1 is -S- and ^X2 is -NH-. The antibody-drug conjugate of any one of claims 1 to 11, wherein ^AK1 is anti-EGFR mAb, anti-Her2 mAb, anti-TWEAKR mAb, anti-CD123 mAb or anti-CXCR5 mAb. The antibody-drug conjugate of any one of claims 1 to 12, wherein ^L1 and ^L2 are each independently: , , , , , , or . The antibody-drug conjugate of any one of claims 1 to 13, wherein ^L1 and ^L2 are each independently: or . The antibody-drug conjugate of any one of claims 1 to 14, wherein each R ¹ is independently fluoro. The antibody-drug conjugate of any one of claims 1 to 15, wherein each R ² is -C(CH ₃ ) ₃ . The antibody-drug conjugate of any one of claims 1 to 16, wherein each R ³ is independently methyl, ethyl, propyl or -C _1-3 alkylene-COOH. The antibody-drug conjugate of any one of claims 1 to 17, wherein each R ³ is independently methyl or -CH ₂ COOH. The antibody-drug conjugate of any one of claims 1 to 18, wherein each R ³ is methyl. The antibody-drug conjugate of any one of claims 1 to 18, wherein each R ³ is -CH ₂ COOH. The antibody-drug conjugate of any one of claims 1 to 20, wherein each R ⁴ is hydrogen or methyl. The antibody-drug conjugate of any one of claims 1 to 21, wherein each R ⁵ is methyl. The antibody-drug conjugate of any one of claims 1 to 22, wherein each R ⁶ is independently fluoro. The antibody-drug conjugate of any one of claims 1 to 23, wherein each R ⁷ is -C(CH ₃ ) ₃ . The antibody-drug conjugate of any one of claims 1 to 24, wherein R ⁸ is methyl, ethyl, propyl or -C _1-3 alkylene-COOH. The antibody-drug conjugate of any one of claims 1 to 25, wherein R ⁸ is methyl or -CH ₂ COOH. The antibody-drug conjugate of any one of claims 1 to 26, wherein R ⁸ is methyl. The antibody-drug conjugate of any one of claims 1 to 26, wherein R ⁸ is -CH ₂ COOH. The antibody-drug conjugate of any one of claims 1 to 28, wherein each R ⁹ is hydrogen or methyl. The antibody-drug conjugate of any one of claims 1 to 29, wherein each R ¹⁰ is methyl. The antibody-drug conjugate of any one of claims 1 to 30, wherein a is an integer from 1 to 25. The antibody-drug conjugate of any one of claims 1 to 31, wherein a is an integer from 1 to 10. The antibody-drug conjugate of any one of claims 1 to 32, wherein b is an integer from 1 to 25. The antibody-drug conjugate of any one of claims 1 to 33, wherein b is an integer from 1 to 10. The antibody-drug conjugate of any one of claims 1 to 34, wherein m is an integer from 1 to 3. The antibody-drug conjugate of any one of claims 1 to 35, wherein m is 2. The antibody-drug conjugate of any one of claims 1 to 36, wherein n is an integer from 1 to 3. The antibody-drug conjugate of any one of claims 1 to 37, wherein n is 2. The antibody-drug conjugate of any one of claims 1 to 38, wherein p is 0, 1 or 2. The antibody-drug conjugate of any one of claims 1 to 39, wherein p is 0 or 1. The antibody-drug conjugate of any one of claims 1 to 40, wherein p is 0. The antibody-drug conjugate of any one of claims 1 to 40, wherein p is 1. The antibody-drug conjugate of any one of claims 1 to 42, wherein q is 0, 1 or 2. The antibody-drug conjugate of any one of claims 1 to 43, wherein q is 0 or 1. The antibody-drug conjugate of any one of claims 1 to 44, wherein q is 0. The antibody-drug conjugate of any one of claims 1 to 44, wherein q is 1. The antibody-drug conjugate of any one of claims 1 to 46, wherein r is 0, 1 or 2. The antibody-drug conjugate of any one of claims 1 to 47, wherein r is 0. The antibody-drug conjugate of any one of claims 1 to 47, wherein r is 1. The antibody-drug conjugate of any one of claims 1 to 49, wherein s is 0, 1 or 2. The antibody-drug conjugate of any one of claims 1 to 50, wherein s is 0 or 1. The antibody-drug conjugate of any one of claims 1 to 51, wherein s is 0. The antibody-drug conjugate of any one of claims 1 to 51, wherein s is 1. The antibody-drug conjugate of any one of claims 1 to 53, wherein the ^RA - ^L1 - ^X1 -fragment is: or . The antibody-drug conjugate of any one of claims 1 to 54, wherein the -X ² -L ^{2 -RB} fragment is: or . The antibody-drug conjugate of any one of claims 1 to 55, wherein the antibody-drug conjugate has a structure of formula (IA), or a pharmaceutically acceptable salt thereof: (IA). The antibody-drug conjugate of claim 56, wherein the antibody-drug conjugate is: , , , , or . The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate has a structure of formula (IB), or a pharmaceutically acceptable salt thereof: (IB). The antibody-drug conjugate of claim 58, wherein the antibody-drug conjugate is: or . The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate has a structure of formula (IC), or a pharmaceutically acceptable salt thereof: (IC). The antibody-drug conjugate of claim 60, wherein the antibody-drug conjugate is: , , or . An antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof: (II) wherein: AK ² is an antibody linked via a sulfur atom or via a nitrogen atom; L ³ is a stabilizing linker; ^RC and ^RD are each independently a protease-cleavable spinodal kinesin inhibitor group; and z is an integer from 1 to 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 62, wherein the antibody-drug conjugate has a structure of formula (II'): (II') wherein: AK ² is an antibody linked via the sulfur atom of cysteine, via the nitrogen atom of lysine, or via a combination thereof; L ³ is a stabilizing linker; P ³ and P ⁴ are each independently a protease-cleavable linker; KSPi ³ and KSPi ⁴ are each independently a spinophilic kinesin inhibitor; and z is an integer between 1 and 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 63, wherein KSPi ³ and KSPi ⁴ are each independently: , or ; wherein: each R ¹¹ is a halogen; and n is an integer from 1 to 5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 63, wherein ^P3 and ^P4 are each independently a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 63, wherein: ^RC is ; R ^D is or ; wherein: each R ¹¹ is a halogen; each R ¹² is a C _1-6 alkyl group or a -C _1-6 alkylene group-CONH ₂ ; each R ¹³ is a C _1-6 alkyl group; each R ¹⁴ is a hydrogen group or a C 1-6 _alkyl group; each R ¹⁵ is a hydrogen group or a C _1-6 alkyl group; each R ¹⁶ is a C _1-6 alkyl group or a -C _1-6 alkylene group-COOH; each R ¹⁷ is a hydrogen group or a C _1-6 alkyl group; n is an integer from 1 to 5; and r, s and t are each independently 0, 1, 2, 3 or 4. The antibody-drug conjugate of any one of claims 62 to 66, wherein ^AK2 is anti-EGFR mAb, anti-Her2 mAb, anti-TWEAKR mAb, anti-mesothelin mAb, anti-CD123 mAb or anti-CXCR5 mAb. The antibody-drug conjugate of any one of claims 62 to 67, wherein ^L3 is , , , , , , , , , , , , or . The antibody-drug conjugate of any one of claims 62 to 68, wherein each R ¹¹ is independently fluoro or chloro. The antibody-drug conjugate of any one of claims 62 to 69, wherein each R ¹¹ is fluoro. The antibody-drug conjugate of any one of claims 62 to 70, wherein each R ¹² is independently methyl, ethyl, propyl or -C _1-3 alkylene-CONH ₂ . The antibody-drug conjugate of any one of claims 62 to 71, wherein each R ¹² is independently methyl or -CH ₂ CONH ₂ . The antibody-drug conjugate of any one of claims 62 to 72, wherein each R ¹² is methyl. The antibody-drug conjugate of any one of claims 62 to 72, wherein each R ¹² is -CH ₂ CONH ₂ . The antibody-drug conjugate of any one of claims 62 to 74, wherein each R ¹³ is independently methyl, ethyl, n-propyl or isopropyl. The antibody-drug conjugate of any one of claims 62 to 75, wherein each R ¹³ is methyl. The antibody-drug conjugate of any one of claims 62 to 75, wherein each R ¹³ is isopropyl. The antibody-drug conjugate of any one of claims 62 to 77, wherein each R ¹⁴ is independently hydrogen, methyl, ethyl or propyl. The antibody-drug conjugate of any one of claims 62 to 78, wherein each R ¹⁴ is independently hydrogen or methyl. The antibody-drug conjugate of any one of claims 62 to 78, wherein R ¹⁴ is hydrogen. The antibody-drug conjugate of any one of claims 62 to 78, wherein R ¹⁴ is methyl. The antibody-drug conjugate of any one of claims 62 to 81, wherein each R ¹⁵ is independently hydrogen, methyl, ethyl or propyl. The antibody-drug conjugate of any one of claims 62 to 82, wherein each R ¹⁵ is methyl. The antibody-drug conjugate of any one of claims 62 to 83, wherein each R ¹⁶ is independently -C _1-6 alkylene-COOH. The antibody-drug conjugate of any one of claims 62 to 84, wherein each R ¹⁶ is independently -C _1-3 alkylene-COOH. The antibody-drug conjugate of any one of claims 62 to 85, wherein each R ¹⁶ is -CH ₂ COOH. The antibody-drug conjugate of any one of claims 62 to 86, wherein each R ¹⁷ is independently C _1-6 alkyl. The antibody-drug conjugate of any one of claims 62 to 87, wherein each R ¹⁷ is independently methyl, ethyl or propyl. The antibody-drug conjugate of any one of claims 62 to 88, wherein each R ¹⁷ is methyl. The antibody-drug conjugate of any one of claims 62 to 89, wherein n is an integer from 1 to 3. The antibody-drug conjugate of any one of claims 62 to 90, wherein n is 2. The antibody-drug conjugate of any one of claims 62 to 91, wherein r is 0, 1 or 2. The antibody-drug conjugate of any one of claims 62 to 92, wherein r is 0 or 1. The antibody-drug conjugate of any one of claims 62 to 93, wherein r is 0. The antibody-drug conjugate of any one of claims 62 to 93, wherein r is 1. The antibody-drug conjugate of any one of claims 62 to 95, wherein s is 0, 1 or 2. The antibody-drug conjugate of any one of claims 62 to 96, wherein s is 0 or 1. The antibody-drug conjugate of any one of claims 62 to 97, wherein s is 0. The antibody-drug conjugate of any one of claims 62 to 97, wherein s is 1. The antibody-drug conjugate of any one of claims 62 to 99, wherein t is 0, 1 or 2. The antibody-drug conjugate of any one of claims 62 to 100, wherein t is 0 or 1. The antibody-drug conjugate of any one of claims 62 to 101, wherein t is 0. The antibody-drug conjugate of any one of claims 62 to 101, wherein t is 1. The antibody-drug conjugate of any one of claims 62 to 103, wherein ^RC is: , , or . The antibody-drug conjugate of any one of claims 62 to 104, wherein ^RD is: , , , , , or . The antibody-drug conjugate of any one of claims 62 to 105, wherein the antibody-drug conjugate is: , , , , , , , , , , , or . An antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt thereof: (III) wherein: AK ³ is an antibody or an antigen-binding fragment thereof linked via a sulfur atom or via a nitrogen atom; L ⁴ is a stabilizing linker; KSP ⁵ is a first spindle-dried kinesin inhibitor; P ⁵ is a protease-cleavable linker; KSP ⁶ is a second spindle-dried kinesin inhibitor; and y is an integer from 1 to 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 107, wherein L ⁴ is a substituted or unsubstituted C _1-60 alkyl linker, or a substituted or unsubstituted 1-60 membered heteroalkyl linker. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 107 or 108, wherein ^L4 has the following structure: or ; where #AK ³ represents the key between L ⁴ and AK ³ ; and # represents the key between L ⁴ and KSPi ⁵ . The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 107 to 109, wherein: KSPi ⁵ is a first spinodal kinesin inhibitor having the following structure: ; KSPi ⁶ is a second spindle-driven kinesin inhibitor having the following structure: or wherein: # represents the bond between L ⁴ and KSPi ⁵ ; ## represents the bond between KSPi ⁵ and P ⁵ ; ### represents the bond between P ⁵ and KSPi ⁶ ; each R ²¹ is independently a halogen; each R ²² is independently a C _1-6 alkyl; and n is an integer from 1 to 5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 107 to 110, wherein ^P5 is a soybean protein cleavable linker comprising one or more of the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-. The antibody-drug conjugate of any one of claims 107 to 110, wherein ^P5 is a soy protein cleavable linker having the following structure: ^-P5a - ^L5 - ^P5b- wherein: ^P5a and ^P5b are each independently a protease cleavable linker; and ^L5 is a stable linker. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 112, wherein ^P5a and ^P5b are each independently a soybean protein cleavable linker selected from the amino acid sequences (1) to (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 112, wherein ^L5 is a stabilizing linker, which is a substituted or unsubstituted _C1-60 alkyl linker, or a substituted or unsubstituted 1-60 membered heteroalkyl linker. The antibody-drug conjugate of any one of claims 107 to 114, wherein ^P5 is a soybean protein cleavable linker having the following structure: , , , or . The antibody-drug conjugate of claim 107 or 115, wherein ^AK3 is anti-EGFR mAb, anti-Her2 mAb, anti-TWEAKR mAb, anti-mesothelin mAb, anti-CD123 mAb or anti-CXCR5 mAb. The antibody-drug conjugate of any one of claims 107 to 116, wherein each R ²¹ is independently fluoro or chloro. The antibody-drug conjugate of any one of claims 107 to 117, wherein each R ²¹ is fluoro. The antibody-drug conjugate of any one of claims 107 to 118, wherein each R ²² is independently C _1-4 alkyl. The antibody-drug conjugate of any one of claims 107 to 119, wherein each R ²² is independently n-butyl, isobutyl, di-butyl or tertiary butyl. The antibody-drug conjugate of any one of claims 107 to 120, wherein each R ²² is a tertiary butyl group. The antibody-drug conjugate of any one of claims 107 to 121, wherein each n is independently an integer from 1 to 3. The antibody-drug conjugate of any one of claims 107 to 122, wherein each n is 2. The antibody-drug conjugate of any one of claims 107 to 123, wherein y is an integer from 1 to 25. The antibody-drug conjugate of any one of claims 107 to 124, wherein y is an integer from 1 to 10. The antibody-drug conjugate of any one of claims 107 to 124, wherein y is an integer from 1 to 6. The antibody-drug conjugate of any one of claims 107 to 126, wherein y is an integer from 2 to 6. The antibody-drug conjugate of any one of claims 107 to 127, wherein: KSPi ⁵ is: ; and KSPi ⁶ is: , or ; Wherein: # represents the key between L ⁴ and KSPi ⁵ ; ## represents the key between KSPi ⁵ and P ⁵ ; and ### represents the key between P ⁵ and KSPi ⁶ . The antibody-drug conjugate of any one of claims 107 to 127, wherein: KSPi ⁵ is ; and KSPi ⁶ is or ; Wherein: # represents the key between L ⁴ and KSPi ⁵ ; ## represents the key between KSPi ⁵ and P ⁵ ; and ### represents the key between P ⁵ and KSPi ⁶ . The antibody-drug conjugate of any one of claims 107 to 129, wherein the antibody-drug conjugate is: , , , , , or . The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 1 to 61, wherein AK is an antibody or an antigen-binding fragment thereof, comprising: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 2; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 3; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 4; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 6; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 7; and/or a light chain complementation determining region 3 comprising the amino acid sequence listed in SEQ ID NO: 8 (LCDR3); b) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 16; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 17; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 18; c) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 16; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 17; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 18; 32; a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 33; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 34; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 36; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 37; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 38; d) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 62; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 63; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 64; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 66; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 67; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 68; e) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 72; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 73; a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 74; a heavy chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 76; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 77; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 78; f) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 82; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 83; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 84; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 77; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 78; f) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 82; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 83; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 84; a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 86; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 87; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 88; g) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 92; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 93; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 94; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 96; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 94; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 96; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 97; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 98; h) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 102; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 103; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 104; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 106; a light chain complement determining region 2 comprising the amino acid sequence listed in SEQ ID NO: 107 (LCDR2); and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence listed in SEQ ID NO: 108; i) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 112; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 113; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 114; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 116; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 117; and/or a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 118; 118; j) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5; k) an immunoglobulin heavy chain variable region comprising an amino acid sequence listed in SEQ ID NO: 1; and/or an immunoglobulin light chain variable region comprising an amino acid sequence listed in SEQ ID NO: 5; l) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 9; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 10 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; m) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 9; and/or an immunoglobulin light chain comprising the amino acid sequence listed in SEQ ID NO: 10; n) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 11; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 15; o) an immunoglobulin heavy chain variable region comprising the amino acid sequence listed in SEQ ID NO: 11; and/or an immunoglobulin light chain variable region comprising SEQ ID NO: 15; p) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 19; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 20; q) an immunoglobulin heavy chain comprising an amino acid sequence listed in SEQ ID NO: 19; and/or an immunoglobulin light chain comprising an amino acid sequence listed in SEQ ID NO: 20; r) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 31; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 35 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain variable region; s) an immunoglobulin heavy chain variable region comprising the amino acid sequence listed in SEQ ID NO: 31; and/or an immunoglobulin light chain variable region comprising the amino acid sequence listed in SEQ ID NO: 35; t) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 39; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 40; u) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 39; and/or an immunoglobulin light chain comprising SEQ ID NO: 40; v) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 61; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 65; w) an immunoglobulin heavy chain variable region comprising an amino acid sequence listed in SEQ ID NO: 61; and/or an immunoglobulin light chain variable region comprising an amino acid sequence listed in SEQ ID NO: 65; x) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 69; and/or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 70 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; y) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 69; and/or an immunoglobulin light chain comprising the amino acid sequence listed in SEQ ID NO: 70; z) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 71; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 75; aa) an immunoglobulin heavy chain variable region comprising the amino acid sequence listed in SEQ ID NO: 71; and/or an immunoglobulin light chain variable region comprising SEQ ID NO: 75; bb) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 79; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 80; cc) an immunoglobulin heavy chain comprising an amino acid sequence listed in SEQ ID NO: 79; and/or an immunoglobulin light chain comprising an amino acid sequence listed in SEQ ID NO: 80; dd) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 81; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 85 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain variable region; ee) an immunoglobulin heavy chain variable region comprising the amino acid sequence listed in SEQ ID NO: 81; and/or an immunoglobulin light chain variable region comprising the amino acid sequence listed in SEQ ID NO: 85; ff) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 89; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 90; gg) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 89; and/or an immunoglobulin light chain comprising SEQ ID NO: 90; hh) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 91; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 95; ii) an immunoglobulin heavy chain variable region comprising an amino acid sequence listed in SEQ ID NO: 91; and/or an immunoglobulin light chain variable region comprising an amino acid sequence listed in SEQ ID NO: 95; jj) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 99; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 100 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; kk) comprising the amino acid sequence listed in SEQ ID NO: 99; and/or comprising the immunoglobulin light chain listed in SEQ ID NO: 100; ll) comprising an immunoglobulin heavy chain variable region with an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 101; and/or comprising an immunoglobulin light chain variable region with an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 105; mm) comprising an immunoglobulin heavy chain variable region with an amino acid sequence listed in SEQ ID NO: 101; and/or comprising SEQ ID NO: 105; nn) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 109; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 110; oo) an immunoglobulin heavy chain comprising an amino acid sequence listed in SEQ ID NO: 109; and/or an immunoglobulin light chain comprising an amino acid sequence listed in SEQ ID NO: 110; pp) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 111; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 115 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain variable region; qq) an immunoglobulin heavy chain variable region comprising the amino acid sequence listed in SEQ ID NO: 111; and/or an immunoglobulin light chain variable region comprising the amino acid sequence listed in SEQ ID NO: 115; rr) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 119; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 120; or ss) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 119; and/or an immunoglobulin light chain comprising SEQ ID NO: An immunoglobulin light chain with the amino acid sequence listed in 120. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 62 to 106, wherein AK ² is an antibody or an antigen-binding fragment thereof, comprising: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 2; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 3; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 4; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 6; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 7; and/or a light chain complementation determining region 3 comprising the amino acid sequence listed in SEQ ID NO: 8 (LCDR3); b) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 12; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 14; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18; c) a heavy chain complementation determining region 1 comprising the amino acid sequence set forth in SEQ ID NO: 32 (HCDR1); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 33; a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 34; a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and/or a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38; d) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 62; a heavy chain complementary determining region 2 comprising the amino acid sequence set forth in SEQ ID NO: 63 (HCDR2); a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 64; a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and/or a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 68; e) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 72; a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 73; a heavy chain complementary determining region 3 comprising the amino acid sequence set forth in SEQ ID NO: 74 (HCDR3); a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 78; f) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 82; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 83; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 84; a light chain complementation determining region 1 comprising the amino acid sequence set forth in SEQ ID NO: 86 (LCDR1); a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88; g) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 92; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 93; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 94; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 96; a light chain complementation determining region 2 comprising the amino acid sequence set forth in SEQ ID NO: 97 (LCDR2); and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 98; h) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 106; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 107; and/or a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 108; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 108; i) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 112; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 113; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 114; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 116; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 117; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 118; j) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1; and/or an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5; k) an immunoglobulin heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 1; and/or an immunoglobulin light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5; l) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 9; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 10; m) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; n) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 11; and/or an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 15; o) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15; p) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 19 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin heavy chain; and / or SEQ ID NO: 20 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; q) comprising the amino acid sequence set forth in SEQ ID NO: 19; and / or comprising the amino acid sequence set forth in SEQ ID NO: 20 immunoglobulin light chain; r) comprising an immunoglobulin heavy chain variable region with an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 31; and / or comprising an immunoglobulin light chain variable region with an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 35; s) comprising SEQ ID NO: 31; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35; t) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 39; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 40; u) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; v) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 61 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin heavy chain variable region; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 65; w) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65; x) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 69; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 70; y) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 69; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 70; z) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 71; and/or an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 75; aa) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 71; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 75; bb) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 79 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin heavy chain; and / or SEQ ID NO: 80 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; cc) comprising the amino acid sequence set forth in SEQ ID NO: 79; and / or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; dd) comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 81; and / or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 85; ee) comprising SEQ ID NO: 81; and/or an immunoglobulin light chain variable region comprising the amino acid sequence listed in SEQ ID NO: 85; ff) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 89; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 90; gg) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 89; and/or an immunoglobulin light chain comprising the amino acid sequence listed in SEQ ID NO: 90; hh) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 90. 91 at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 91; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 95; ii) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95; jj) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 99; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 100; kk) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; ll) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 101; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 105; mm) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105; nn) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 101; 109 at least 85%, 90%, 95%, 97%, 98% or 99% identical to an immunoglobulin heavy chain; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 110; oo) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 109; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 110; pp) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 111; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 115; qq) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; rr) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 119; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 120; or ss) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 119; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 120. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 107 to 130, wherein AK ³ is an antibody or an antigen-binding fragment thereof, comprising: a) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence listed in SEQ ID NO: 2; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence listed in SEQ ID NO: 3; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence listed in SEQ ID NO: 4; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence listed in SEQ ID NO: 6; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence listed in SEQ ID NO: 7; and/or a light chain complementation determining region 3 comprising the amino acid sequence listed in SEQ ID NO: 8 (LCDR3); b) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 12; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 14; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18; c) a heavy chain complementation determining region 1 comprising the amino acid sequence set forth in SEQ ID NO: 32 (HCDR1); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 33; a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 34; a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and/or a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38; d) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 62; a heavy chain complementary determining region 2 comprising the amino acid sequence set forth in SEQ ID NO: 63 (HCDR2); a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 64; a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and/or a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 68; e) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 72; a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 73; a heavy chain complementary determining region 3 comprising the amino acid sequence set forth in SEQ ID NO: 74 (HCDR3); a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 78; f) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 82; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 83; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 84; a light chain complementation determining region 1 comprising the amino acid sequence set forth in SEQ ID NO: 86 (LCDR1); a light chain complementation determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and/or a light chain complementation determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88; g) a heavy chain complementation determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 92; a heavy chain complementation determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 93; a heavy chain complementation determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 94; a light chain complementation determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 96; a light chain complementation determining region 2 comprising the amino acid sequence set forth in SEQ ID NO: 97 (LCDR2); and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 98; h) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 106; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 107; and/or a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 108; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 108; i) a heavy chain complement determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 112; a heavy chain complement determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 113; a heavy chain complement determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 114; a light chain complement determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 116; a light chain complement determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 117; and/or a light chain complement determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 118; j) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1; and/or an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5; k) an immunoglobulin heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 1; and/or an immunoglobulin light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5; l) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 9; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 10; m) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; n) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 11; and/or an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 15; o) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15; p) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 19 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin heavy chain; and / or SEQ ID NO: 20 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; q) comprising the amino acid sequence set forth in SEQ ID NO: 19; and / or comprising the amino acid sequence set forth in SEQ ID NO: 20 immunoglobulin light chain; r) comprising an immunoglobulin heavy chain variable region with an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 31; and / or comprising an immunoglobulin light chain variable region with an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 35; s) comprising SEQ ID NO: 31; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35; t) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 39; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 40; u) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; v) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 61 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin heavy chain variable region; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 65; w) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65; x) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 69; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 70; y) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 69; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 70; z) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 71; and/or an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 75; aa) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 71; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 75; bb) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 79 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin heavy chain; and / or SEQ ID NO: 80 at least 85%, 90%, 95%, 97%, 98% or 99% identical to the immunoglobulin light chain; cc) comprising the amino acid sequence set forth in SEQ ID NO: 79; and / or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; dd) comprising an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 81; and / or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 85; ee) comprising SEQ ID NO: 81; and/or an immunoglobulin light chain variable region comprising the amino acid sequence listed in SEQ ID NO: 85; ff) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 89; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 90; gg) an immunoglobulin heavy chain comprising the amino acid sequence listed in SEQ ID NO: 89; and/or an immunoglobulin light chain comprising the amino acid sequence listed in SEQ ID NO: 90; hh) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 90. 91 at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 91; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 95; ii) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95; jj) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 99; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 100; kk) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; ll) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 101; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 105; mm) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105; nn) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 101; 109 at least 85%, 90%, 95%, 97%, 98% or 99% identical to an immunoglobulin heavy chain; and/or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 110; oo) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 109; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 110; pp) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 111; and/or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 115; qq) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; and/or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; rr) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 119; and/or an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 120; or ss) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 119; and/or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 120. A pharmaceutical composition comprising at least one antibody-drug conjugate according to any one of claims 1 to 133 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable formulation. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 133 or a pharmaceutically acceptable salt thereof, and a pharmaceutically suitable inert non-toxic auxiliary agent. A method for treating a disease, comprising administering the antibody-drug conjugate of any one of claims 1 to 133 to a patient in need thereof. A method for treating a hyperproliferative and/or angiogenic disorder, the method comprising administering to a patient in need thereof the antibody-drug conjugate of any one of claims 1 to 133, or the pharmaceutical composition of claim 134.