Conjugates Containing Phosphoantigens and Uses in Therapy - Patent application

Abstract

The present invention relates to novel conjugates comprising a targeting moiety linked to a phosphoantigen and their use, optionally in combination with other therapeutic agents, in the treatment of diseases, such as cancer, infectious diseases and autoimmune diseases. The targeting moiety may be an antibody or a binding fragment thereof. The phosphoantigen may be a prodrug. Furthermore, the present invention relates to linker-drug compounds comprising the phosphoantigen for use in the preparation of the conjugates and pharmaceutical compositions comprising said immunoconjugates.

Description

[Technical field]

[0001] The present invention relates to novel conjugates comprising a targeting moiety, such as an antibody or a binding fragment thereof, linked to a phosphoantigen, and their use, optionally in combination with other therapeutic agents, in the treatment of diseases such as cancer, infectious diseases and autoimmune diseases.Furthermore, the present invention relates to linker-drug compounds comprising the phosphoantigen for use in preparing said conjugates and pharmaceutical compositions comprising said immunoconjugates. [Background technology]

[0002] The usual methods for treating cancer include surgery, chemotherapy with cytotoxic agents and radiation therapy or a combination thereof. Treatment with cytotoxic agents or radiation often leads to severe side effects due to its toxicity and non-specific nature. As it has been discovered that the immune system plays an important role in eradicating neoplastic cells, recent cancer treatments aim to use components of the immune system as a tool to treat cancer.

[0003] One approach to cancer immunotherapy is to target "immune checkpoints", e.g., T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1), aiming to activate antitumor immune responses in cancer patients. Both CTLA-4 and PD-1 are proteins involved in a negative feedback system that functions to limit the activation of immune cells. Tumor cells can "exploit" this inhibitory mechanism by overexpressing immune checkpoint ligands on their surface to protect themselves from attack by cells of the immune system and evade the immune system. Activation of immune checkpoints through interaction with their ligands leads to inactivation and exhaustion of T cells. Immune checkpoint inhibitors, e.g., antibodies that bind to immune checkpoints or their ligands, are new anticancer drugs that block immune checkpoints overexpressed on cancer cells. Examples of approved immune checkpoint inhibitors are ipilimumab (blocks CTLA-4; marketed under the trade name Yervoy® and manufactured by BMS), approved in 2011 for the treatment of melanoma, the PD-1 antibody nivolumab (sold under the trade name Opdivo® and developed by BMS), and pembrolizumab (marketed under the trade name Keytruda®, another PD-1 inhibitor manufactured by Merck). Checkpoint inhibitors can reinvigorate anti-tumor responses, but activated immune cells can also attack normal tissues, resulting in adverse immunological side effects.

[0004] Another approach to cancer therapy involves the use of antibody-drug conjugates (ADCs). ADCs combine the specificity of monoclonal antibodies for tumor-specific antigens with the cell-killing activity of chemical cytotoxic agents. The antibody of the ADC serves as a targeting agent and a carrier of the cytotoxic payload. Once the antibody binds to the target, the ADC conjugated with the cytotoxic payload is efficiently taken up by the targeted tumor cells. The cytotoxic payload may be an inactive precursor (prodrug) of the cytotoxic agent grafted onto the antibody via a linker that is stable in the blood and cleaved, for example, by intracellular proteases after internalization of the tumor cells. Cleavage of the linker may cause the release of the active cytotoxic form of the payload in the tumor cells. ADCs have the advantage of significantly reducing toxicity to healthy tissues and non-specific side effects. Clinically approved ADCs include gemtuzumab (anti-CD33) ozogamicin (Mylotarg®; Wyeth Pharmaceuticals, a subsidiary of Pfizer), brentuximab (anti-CD30) vedotin (Adcetris®; Seattle Genetics / Millennium Pharmaceuticals), (ad-)trastuzumab (anti-HER2) emtansine (Kadcyla®; Genentech / Roche), inotuzumab (anti-CD22) ozogamicin (Besponsa®; Wyeth Pharmaceuticals, a subsidiary of Pfizer), enfortumab (anti-nectin-4) vedotin (Padosev™; Astellas Pharma / Seattle Genetics), and fam-trastuzumab deruxtecan (Enhertz®; Daiichi These include polatuzumab (anti-CD79b) vedotin (Poraivy™; Genentech / Roche), and sacituzumab (anti-TROP-2) govitecan (Trodelvy™; Immunomedics). Many more are in clinical development.

[0005] Yet another approach to cancer treatment is immunotherapy, which uses therapeutic compounds to activate the immune system, particularly T cells, to attack and destroy tumor cells. Such therapeutic compounds may be agonists of immune cell receptors and may be large molecules or relatively small chemical structures. Examples of such compounds are ligands that activate Toll-like receptors (TLRs). Several TLR ligands have been approved for the treatment of cancer. The first approved TLR ligand (TLR agonist) was part of an attenuated strain of Mycobacterium bovis called Bacillus Calmette-Guerin (BCG). Initially developed as a tuberculosis vaccine, BCG contains active TLR2 / 4 ligands and has been used to treat bladder cancer. Other approved TLR ligands are the TLR4 ligand monophosphoryl lipid A (MPLA) and the small molecule TLR7 agonist imiquimod, an imidazoquinoline.

[0006] TLR ligands have also been used as immunoconjugates. Such immunoconjugates contain antibodies specific for tumor antigens as targeting vehicles for TLR ligands to induce local activation of immune system cells in the tumor microenvironment. Immunoconjugates for treating breast cancer, in which TLR agonists are linked to anti-HER antibodies, are described in WO 2017 / 072662 (Novartis AG). Anti-HER conjugates with a TLR8 agonist payload have been developed by Silverback Therapeutics (ImmunoTAC™ SBT6050). Bolt Therapeutics (WO 2020 / 047187) and Ackerman et al., 2021, Nature Cancer, , Vol. 2(8), 18-33 also describe TLR immunoconjugates comprising a tumor-targeting monoclonal antibody conjugated via a non-cleavable linker to a TLR7 / 8 agonist (T785); the tumor-targeting antibody bound to the tumor antigen activates antigen-presenting cells present in the tumor microenvironment (TME) by Fc effector function, while the TLR agonist conjugated to the antibody directly stimulates APCs via their TLR receptors, thereby promoting anti-tumor immunity.

[0007] A particular subset of T cells known to exhibit cytotoxicity against cancer cells are γδ T cells (T cells with a T cell receptor (TCR) composed of γ and δ chains). γδ T cells are considered a unique subset of T lymphocytes due to their ability to mount a rapid innate immune response to infection and tumor cells. Tumor-infiltrating γδ T cells have been found in many different malignancies (Gentles et al., Nature Medicine, 2015, 21(8), 938-945). γδ T cells, or more specifically, Vγ9Vδ2 T cells, which form the major subset of γδ T cells, are activated by a specific set of antigens known as "phosphoantigens". Naturally occurring phosphoantigens are small alkyl pyrophosphates, such as 4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP). These natural phosphoantigens are produced by pathogenic cells, and HMBPP is the direct precursor of IPP (HMBPP is a pathogenic phosphate antigen that is not present in humans). Bacteria and parasites produce isoprenoid precursors via the non-mevalonate pathway (MEP) or 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP / DOXP) pathway, resulting in the biosynthesis of the isoprenoid precursor IPP. In humans, pAg production is driven by the mevalonate pathway.

[0008] In contrast to TLR agonists, phosphoantigens do not directly interact with receptors presented on myeloid or T cells. Intracellular (e.g., cancer cells) binding of phosphoantigens to the intracellular domain of the cell surface molecule butyrophilin 3A1 (BTN3A1) is thought to induce a conformational change in the extracellular portion of the BTN3A1 complex, as well as BTN2A1 (Sandstrom A, et al., 2014, Immunity, 40(4), 490-500, doi: 10.1016 / j.immuni.2014.03.003).

[0009] The conformational change of the extracellular BTN3A1 / BTN2A1 complex results in binding to the γδTCR, leading to cytokine production and tumor / pathogenic cell killing by the activated γδT cells (Rigau et al., Science, 2020, 367, 642). Thus, activation of γδT cells using phosphoantigens as therapeutic agents is indirect; phosphoantigens act from within a cell (e.g., a tumor cell or an infected cell) to change the conformation of the extracellular BTN3A1 / BTN2A1 complex on the cell surface, which then transmits an activation signal to the γδTCR on the γδT cell. The γδT cell then exerts a cell killing effect on the tumor cell or infected cell.

[0010] Pyrophosphate HMBPP has poor pharmacokinetic properties (rapid hydrolysis in plasma), so (nitrogenous) bisphosphonate analogs as well as (monophosphate) prodrug forms (converted to active phosphoantigens after administration to a subject) have been developed. In phosphoantigen prodrugs, the negatively charged unbound oxygen atom of the phosphonate group is protected with a neutral group, for example to increase diffusion into the cell membrane. The protecting group is removed once inside the cell, releasing the active phosphoantigen. Another approach to improve the half-life of phosphoantigens (particularly bisphosphonate phosphoantigens) is described in WO 2012 / 042024. Phosphoantigens were complexed with inorganic nanoparticles and lipid nanovectors that act as delivery vehicles. It was proposed that the resulting nanoparticles could be coated with targeting ligands on their surface to target specific cells. Examples include molecules that induce targeting to cancer cells, such as antibodies. The use of human transferrin was exemplified.

[0011] Phosphoantigens have been tested for use in cancer therapy, aiming to improve the cytotoxicity of γδ T cells to tumor cells by expanding γδ T cells in vivo or in vitro with antigen-presenting cells for administration to subjects. Synthetic phosphoantigens such as BrHPP (Phosphostim, manufactured by Innate Pharma) and Zoledronate (Novartis) are the subject of clinical trials in cancer patients. The phosphoantigens tested have shown acceptable safety profiles. However, their efficacy has generally been lacking (Sebestyen et al., Nature Reviews Drug Discovery, 2020,19(3), 169-184).

[0012] Finding an acceptable therapeutic time window / therapeutic concentration window for such treatments could be greatly improved by more potent, selective and effective methods for delivering phosphoantigens to cells (over)expressing the butyrophilin (BTN3A1 / BTN2A1) complex, e.g. tumor cells or pathogenic cells. Summary of the Invention

[0013] The present invention provides more effective and selective methods of using phosphoantigens, for example in the treatment of cancer. The present invention relates to a conjugate comprising a targeting moiety covalently linked to an immunomodulatory moiety, where the immunomodulatory moiety is a phosphoantigen (pAg). Preferably, the targeting moiety is a tumor-targeting antibody or an antigen-binding fragment thereof. Such conjugates can be used to activate γδ T cells in the treatment of diseases such as cancer, infectious diseases, and autoimmune diseases. The conjugates of the invention can be used alone or in combination with other therapeutic agents.

[0014] Preferably, the conjugate of the present invention is an immunoconjugate comprising a tumor-targeting antibody or an antigen-binding fragment thereof as a targeting moiety. The conjugate of the present invention comprising a tumor-targeting antibody as a targeting moiety can be used to specifically deliver phosphoantigens to localized tumor cells, and they may be internalized within the tumor cells after the antibody or antigen-binding fragment thereof binds to a tumor-specific or tumor-associated antigen (TAA). The present invention also provides a linker-drug compound for use in the preparation of the conjugate of the present invention, wherein the "drug" is a phosphoantigen. Furthermore, the present invention provides a pharmaceutical composition comprising the conjugate of the present invention and one or more pharmaceutical additives. The conjugate of the present invention may be used, for example, as a medicament for the treatment of cancer. [Brief description of the drawings]

[0015] [Figure 1] FCC / gating strategy to identify different immune cell populations. First, a time gate was applied to ensure constant flow (A). Subsequently, lymphocyte doublets were excluded on FSC-A vs. FSC-H plots (B) and SSC-A vs. SSC-H plots (C). Viable cells were then selected (D) and subsequently lymphocytes were selected (E). CD3-negative CD56-positive cells were then identified as NK cells (F). CD3-positive cells were divided into Vd2-positive and Vd1-positive cells (G). CD3-positive Vd2-negative Vd1-negative lymphocytes were also subdivided into CD8-positive cytotoxic T cells (H). [Diagram 2] Production of CD107a (Figure 2A) or IFNγ (Figure 2C) by Vδ2γδ T cells. Levels of activation are shown as the percentage of Vδ2γδ T cells that are IFNγ+ or CD107a+. Measurements were performed in four healthy donors and are shown for the Vδ2γδ T cell population for all donors. (In some graphs in Figures 2 and 4, "IFNγ" is written as "IFNy"). For one representative donor, CD107a+ production (Figure 2B) and IFNγ production (Figure 2D) are shown for NK cells, Vδ1 γδ T cells, or CD8+ T cells gated from total PBMCs co-cultured with Raji cells pre-treated with various concentrations of prodrug / HMBPP(M). [Diagram 3] CD107a or IFNγ production by PHA-activated V52 γδ T cells (A,I), NK cells (B,J), V51 γδ T cells (C,K) and CD8+ T cells (D,L) or unstimulated V52 γδ T cells (E,M), NK cells (F,N), V51 γδ T cells (G,O) and CD8+ T cells (H,P). FACS plots show CD107a (A-H) or IFNγ (I-P) profiles of representative healthy donors. [Figure 4] CD107a (A-D) and IFNγ (E-H) production by gated Vδ2 γδ T cells (A,E), NK cells (B,F), Vδ1 γδ T cells (C,G), and CD8+ T cells (D,H) after co-culture of PBMCs with Raji cells pretreated with various concentrations of ADC or rituximab. Levels of activation are shown as percentages of IFNγ+ or CD107a+ immune cell subsets. Measurements were performed in three (ADC-XD13-r, ADC-XD13-i) or four (ADC-XC4-r, ADC-XC4-i, ADC-XD4-r, ADC-XD4-i, rituximab) healthy donors. Results are shown for one representative donor. [Diagram 5] EC50 (A-B), maximum percentage of Vδ2γδ T cells positive (C-D), and median fluorescence intensity (MFI, E-F) for CD107a (A, C, E) or IFNγ (B, D, F) production of Vδ2γδ T cells after co-culture with ADC- or rituximab-treated Raji cells. MFI was determined for CD107a+ or IFNγ+ Vδ2γδ T cells. Measurements were performed in three (ADC-XD13-r) or four (ADC-XC4-r, ADC-XD4-r, rituximab) healthy donors and each symbol represents an individual donor. [Figure 6] Representative IFNγ / CD107a profiles of electronically gated Vδ2γδ T cells cocultured with ADC-XC4-r (A), ADC-XD4-r (B), ADC-XD13-r (C), and rituximab (D) pretreated Raji cells. [Figure 7]Direct effect of rituximab, zoledronate, HMBPP, pAg conjugates or duocarmycin as positive control on Raji cell viability measured after 6 days of incubation. Duocarmycin was used as a positive control. (A) Different concentrations of compounds were incubated with Raji cells and cell death was confirmed. Control and free drug are shown in one graph, different pAg conjugates are separated into three graphs for ease of reading. (B) Summary of cell viability (%) for highest pAg conjugates or rituximab (concentration 10 μg / mL). [Figure 8] CD107a production by gated Vδ2 γδ T cells after co-culture of PBMCs with Raji cells pretreated with different concentrations of pAg complexes or rituximab. The level of activation is shown as the percentage of immune cell subsets that are CD107a+. Measurements were performed in healthy donors. Each plot represents a different experiment and each letter indicates the donor used. If the same donor was tested in two different experiments, it is indicated with "-1" or "-2". [Figure 9] CD107a (A, D, G), IFNγ (B, E, H) and TNFα (C, F, I) production by gated Vδ2 γδ T cells after co-culture of PBMCs with Raji cells pretreated with various concentrations of pAg conjugates or rituximab. Box plots show EC50 (A, B, C), % cell activation (D, E, F) or median fluorescence intensity (MFI, G, H, I). Dots in box plots (D-I) represent mean values. Graphical representation of Tables 5-10. Note that TNFα production was not investigated in all experiments. [Figure 10] Correlation between CD107a and IFNγ production (EC50, % activity, and MFI) by γδ T cells co-cultured with Raji cells pretreated with pAg conjugates. The activity of pAg conjugates was tested in different experiments with different donors and the geometric mean EC50 (A) or mean activation rate (B) and mean "median fluorescence intensity" (MFI, C) were calculated for each pAg conjugate. Correlations between EC50, activation rate, and MFI of CD107a and IFNγ are plotted. Rituximab is shown as a dotted line. [Figure 11]EC50 (A–C) and maximum percentage (D–F) of positive Vδ2γδ T cells for CD107a (A,D), IFNγ (B,E) and TNFα (C,F) after co-culture with Raji cells pretreated with ADC-XD65-r, ADC8-XD65-r or rituximab. Measurements were performed in two healthy donors. [Figure 12] Killing of Raji cells pretreated with pAg complexes by V52 γδ T cells. Raji cells were pretreated for 16 h with no compound (effector + target; E+T; grey squares), HMBPP (+ symbols) or with various concentrations of ADC-XD18-r (black circles), ADC-XD18-i (open circles, dotted lines) or rituximab (grey diamonds) and subsequently co-cultured with expanded V52 γδ T cells for 1 h. Killing of Raji cells was then assessed using flow cytometry. (A) Dose-dependent killing of Raji cells by donor-derived expanded V52 γδ T cells. (B) Correlation between rituximab potency and CD16 expression on expanded V52 γδ T cells. (C, D) Summary graphs show the EC50 (C) and potency (D) of the percentage of dead Raji cells for all donors tested. [Figure 13] Activity of ADC-XD18-i, ADC-XD18-r, rituximab and HMBPP against Vδ2γδ T cells after pretreatment with multiple CD20 positive cell lines. (A) Percentage of CD107a positive Vδ2γδ T cells after co-culture with cell lines pretreated with HMBPP. (B-F) Percentage of CD107a positive Vδ2γδ T cells after co-culture with cell lines pretreated with various concentrations of ADC-XD18-i, ADC-XD18-r or rituximab. All experiments were performed in two healthy donors as indicated on the graph. [Figure 14]Percentage of Vδ2γδ T cells producing CD107a (A-D), IFNγ (E-H) or TNFα (I-L) after coculture with BT-474 (A,E,I), SK-BR-3 (B,F,J), SK-OV-3 (C,G,K) or HCT-116 cells (D,H,L) pretreated with various concentrations of ADC-XD18-t, ADC-XD18-i or trastuzumab. Results are shown for one representative donor from two individuals (BT-474, SK-OV-3, and HCT-116) and four individuals (SK-BR-3). [Figure 15] EC50 (A–C) and potency (D–F) of CD107a (A,D), IFNγ (B,E) or TNFα (C,F) production of Vδ2 γδ T cells after 6 h of co-culture with cell lines treated with pAg conjugates or trastuzumab. Each symbol represents a healthy donor and shows the geometric mean (A–C) or mean (D–F). [Figure 16] Percentage of CD107a positive (A), IFNγ positive (B) or TNFα positive (C) Vδ2γδ T cells after 6 h of co-culture with cell lines pretreated with HMBPP. Each symbol represents an individual healthy donor and the mean is shown. [Figure 17] Percentage of CD107a-producing NK cells after co-culture with BT-474, SK-BR-3, SK-OV-3 or HCT-116 cells pretreated with various concentrations of ADC-XD18-t, ADC-XD18-i or trastuzumab. Results are shown for one representative donor from two (BT-474, SK-OV-3 and HCT-116) and four (SK-BR-3). [Figure 18]CD107a production by gated Vδ2γδ T cells after co-culture of PBMCs with Raji or MOLM-13 cells pretreated with different concentrations of pAg conjugates or control compounds. (A) % activity (CD107a) of Vδ2γδ T cells after co-culture with HMBPP pre-treated Raji or MOLM-13 cells. (B-E) Levels of activation of Vδ2γδ T cells in PBMCs derived from different healthy donors after co-culture with pre-treated Raji (top) or MOLM-13 cells (bottom). Each plot represents a different experiment and each letter indicates a donor. (F-H) Summary graphs show EC50 (F), potency (G) and median fluorescence intensity (MFI, H) for all donors tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention The present invention provides a conjugate comprising a targeting moiety covalently linked to an immunomodulatory moiety, where the immunomodulatory moiety is a phosphoantigen (pAg) moiety.

[0017] Complex The present invention provides a conjugate comprising a targeting moiety covalently linked to an immunomodulatory moiety, where the immunomodulatory moiety is a phosphoantigen (pAg) moiety.

[0018] The conjugates of the present invention comprise a targeting moiety that specifically binds to a target cell. Preferably, the targeting moiety is a tumor-targeting antibody or an antigen-binding fragment thereof. The targeting moiety serves as a delivery vehicle; it delivers the pAg, which is covalently linked to the targeting moiety, to the target cell. The pAg may be directly linked, for example, to an amino acid side chain in the (polypeptide) targeting moiety. Preferably, the pAg is conjugated to the targeting moiety via a linking moiety.

[0019] Preferred conjugates of the present invention have the general formula I: Tm-(L-(pAg) x ) y (I) where Tm represents a targeting moiety, preferably an antibody or antigen-binding fragment thereof, L represents a linking moiety, pAg represents a phosphoantigen, x represents the number of phosphoantigens per linking moiety and is 1 to 5, and y is the ratio of L-(pAg) per Tm. x (represents the average number of linker moieties per targeting moiety) and is an integer from 1 to 10, preferably an integer from 1 to 8) The number of pAg per conjugate in formula I (ratio of pAg to Tm) is x multiplied by y. The average ratio of pAg to Tm may be from 1 to 16 or 20 or more. The ratio of pAg units per targeting moiety may vary, for example, depending on the structural or functional features of either the phosphoantigen or the targeting moiety. In fact, even a number of pAg per targeting moiety as low as 2-8 or 2-6, or even 2, may provide sufficient therapeutic effect. Preferably, the linking moiety has one or two pAgs. In most cases, one pAg per linking moiety may be sufficient. Preferably, the ratio of target pAg to Tm is 2 (x is 1 and y is 2).

[0020] The linker moiety is preferably a cleavable linker moiety. Linear or branched linker moieties may be used in the conjugates of the invention. When multiple phosphoantigens are attached to one targeting moiety, each phosphoantigen may be covalently attached to the targeting moiety by a separate linking moiety. Indeed, when the targeting moiety is an antibody and the linking occurs with reduced interchain disulfides, there may be at most eight separate linking moieties attached to one targeting moiety, resulting in eight phosphoantigens per targeting moiety when each phosphoantigen is carried by its own linking moiety. Another branched linker moiety may contain 1-5 phosphoantigens (x is 1, 2, 3, 4 or 5) per linking moiety. Branched linkers are preferred, especially when a high ratio of pAg to Tm is desired or when the available binding sites on the targeting moiety are limited. For example, a branched linker with two pAgs (x is 2) can be used to increase the number of phosphoantigens per targeting moiety. By using such linking moieties, for example, 16 phosphoantigens can be linked to the targeting moiety with only 8 linking moieties. The antibody can be modified to add cysteines in its amino acid sequence that can form disulfide bonds and be reduced to conjugate with the linker-drug molecule. For example, an additional cysteine ​​can be introduced, for example, at position 41C, as disclosed in WO 2015 / 177360. An antibody with up to 10 cysteines available for conjugation to which a linking moiety can be attached can have a DAR of 20 (x is 2 and y is 10) or more when a branched linker with 2 pAgs (x is 2) per linker is used. Under optimal conditions, all binding sites on the targeting moiety are occupied by linking moieties. In practice, a mixture of conjugates may be produced, in which case the exact number of phosphoantigens per targeting moiety may vary slightly depending on the reaction conditions, and the y value is an average number.

[0021] The conjugates of the invention comprising phosphoantigens may be used in combination with other pharma- ceutical active compounds that can be administered simultaneously or sequentially to a subject in need thereof. Furthermore, the targeting moiety may carry a combination of phosphoantigens and different payloads. The advantage of such a "multiple payload" approach is that different active substances are targeted by the same targeting moiety. The reaction conditions (of conjugation) and the binding sites as well as the ratio between the payloads must be set appropriately. Separate linker-drug compounds for each payload may be conjugated, for example, at different binding sites (e.g. different types of amino acids) on the targeting moiety and / or by different conjugation methods and / or linkers of different chemical nature to adjust the binding, distribution and drug-antibody ratio (DAR) of the different payloads. Antibody-drug conjugates (ADCs) with multiple cytotoxic payloads are known in the art. The conjugates of the invention may combine phosphoantigens with, for example, a cytotoxic payload or another immunomodulatory payload designed to enhance the overall desired therapeutic effect. Thus, non-specific binding and / or effects of phosphoantigens at non-target sites on non-target tissues are reduced.

[0022] As is well known in the art, binding of a drug in an ADC can be determined, for example, by hydrophobic interaction chromatography (HIC) or reverse phase high performance liquid chromatography (RP-HPLC). HIC is particularly suitable for determining the average DAR (ratio of pAg to Tm in the conjugates of the invention).

[0023] Targeting section The targeting moiety specifically or preferentially binds to the target cell and may be a targeting antibody or an antigen-binding fragment thereof, or another targeting moiety such as a nucleic acid (aptamer) or a (poly)peptide, which may be an enzyme inhibitor, an enzyme substrate, a receptor ligand and / or a fusion protein. Small molecule inhibitors can also be used as targeting moieties (resulting in small molecule drug conjugates (SMDCs)). The binding specificity (and affinity) of the targeting moiety for the target determines the site in the body where the conjugate of the invention exerts its therapeutic effect.

[0024] Thus, selection of an appropriate targeting moiety ensures that the phosphoantigen is delivered to the site where it must exert its therapeutic effect.

[0025] Preferably, the targeting moiety in the conjugate of the invention is an antibody or an antigen-binding fragment thereof. When the targeting moiety is an antibody or an antigen-binding fragment thereof, the conjugate is generally called an immunoconjugate or an antibody-drug conjugate (ADC). The targeting moiety is an antibody that recognizes with high specificity an antigen expressed in the target cell, e.g., a tumor-associated antigen. The specificity of the antibody or its fragment for the antigen allows the specific delivery of the effector molecule (or "payload") to the target cell, with little effect on healthy tissue. The effector molecule is covalently attached to the antibody via a linker, which ensures that the effector molecule remains connected to the antibody at least until the antibody reaches the target cell, e.g., a cancer cell. The effector molecule exerts its effect on or in the target cell (when the conjugate is internalized) when the antibody binds to the target. The effector molecule may be a cytotoxic agent, a radioisotope, or an immunomodulatory moiety. In the conjugate of the invention, the effector molecule is a phosphoantigen.

[0026] antibody In this specification, the term "antibody" preferably refers to an antibody comprising two heavy chains and two light chains. Generally, an antibody or an antigen-binding fragment thereof has therapeutic activity, but such an independent effect is not necessarily required, as known in the field of ADCs. The antibody that can be used in the present invention may be of any isotype, for example, an IgA, IgE, IgG or IgM antibody. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG2 antibody. The antibody may be a chimeric, humanized or human antibody. Preferably, the antibody is a humanized or human antibody. Even more preferably, the antibody is a humanized or human IgG antibody, more preferably a humanized or human IgG1 monoclonal antibody. The antibody may have a κ or λ light chain, preferably a κ light chain, i.e., a humanized or human IgG1-κ antibody.

[0027] As used herein, the term "antigen-binding fragment" includes Fab, Fab', F(ab')2, Fv, scFv, or reduced IgG (rIgG) fragments, single-chain (sc) antibodies, single domain (sd) antibodies, diabodies, or minibodies.

[0028] A "humanized" non-human (e.g. rodent) antibody is an antibody that contains minimal sequence derived from a non-human antibody (e.g. a non-human-human chimeric antibody). Various methods for humanizing non-human antibodies are known in the art. For example, the antigen-binding complementarity determining regions (CDRs) in the variable regions (VRs) of the heavy chain (HC) and light chain (LC) are derived from an antibody from a non-human species, typically mouse, rat or rabbit. These non-human CDRs are combined with human framework regions (FRs, i.e. FR1, FR2, FR3 and FR4) of the HC and LC variable regions such that the functional properties of the antibody, e.g. binding affinity and specificity, are at least partially retained. To further refine the antibody performance, for example to improve binding affinity while retaining low immunogenicity, selected amino acids of the human FRs may be exchanged with the corresponding amino acids of the original non-human species. The variable regions thus humanized are usually combined with human constant regions. A representative method for humanizing non-human antibodies is that of Winter and coworkers (Jones et al, 1986, Nature, 321, 522-525; Riechmann et al, 1988, Nature, 332, 323-327; Verhoeyen et al, 1988, Science 239, 1534-1536). Alternatively, non-human antibodies can be humanized by modifying their amino acid sequence to increase similarity to antibody variants naturally produced in humans. For example, selected amino acids of the original non-human species FRs can be replaced with their corresponding human amino acids to reduce immunogenicity while retaining the binding affinity of the antibody. For details, see Jones et al, supra; Riechmann et al., supra and Presta, 1992, Curr. Op. Struct. Biol. 2, 593-596. See also the following reviews and references cited therein:Vaswani and Hamilton, 1998, Ann. Allergy, Asthma and Immunol., 1, 105-115; Harris, 1995, Biochem. Soc. Transactions, 23, 1035-1038 and Hurle and Gross, 1994, Curr. Op. Biotech., 5, 428-433.

[0029] CDRs can be determined by the method of Kabat (in Kabat, EA et al, (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, NIH publication no. 91-3242, pp. 662, 680, 689), Chothia ( et al, 1989, Nature, 342, 877-883) or IMGT (Lefranc, 1999, The Immunologist, 7, 132-136).

[0030] Typically, the antibodies are monospecific (specific for one antigen; such antigens may be common between species or may have similar amino acid sequences between species) or bispecific (specific for two different antigens in a species) antibodies that contain at least one HC and LC variable region that binds to an antigen target, preferably a membrane-bound antigen target that may or may not be internalized. Preferably, after binding to the (antigen) target, the antibody is internalized into the target cell, after which the active effector molecule, which in the complex of the invention is a phosphoantigen, is released into the cell.

[0031] The targeting antibodies that may be used in the conjugates of the invention for use in cancer therapy may be tumor-targeting antibodies that selectively bind to tumor-specific or tumor-associated antigens. Tumor-specific antigens are present only on tumor cells, whereas tumor-associated antigens are antigens that are expressed at higher levels (e.g., overexpressed) in cancer cells when compared to normal (healthy) cells.

[0032] Antigen targets to which the antibodies or antigen-binding fragments of the conjugates of the invention bind include, for example, annexin A1, B7H3, B7H4, BCMA, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242 (cancer antigen 242), CAIX, CCR2, CCR5, CD2, CD19, CD20, CD22, CD24, CD30 (tumor necrosis factor 8), CD33, CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44, CD47 (integrin-associated protein), CD56 (neural cell adhesion molecule), CD70, CD71, CD73, CD74, CD79, CD115 (colony-stimulating factor 1 receptor), CD123 (interleukin-3 receptor), CD138 (syndecan-1), CD203c (ENPP3), CD303, CD333, CDCP1, CEA, CEACAM, claudin 4, claudin 7, CLCA-1 (C-type lectin-like molecule-1), CLL 1, c-MET (hepatocyte growth factor receptor), Cripto, DLL3, EGFL, EGFR, EPCAM, EphA2, EPhB3, ETBR (endothelin type B receptor), FAP, FcRL5 (Fc receptor-like protein 5, CD307), FGFR3, FOLR1 (folate receptor α), FRbeta, GCC (guanylyl cyclase C), GD2, GITR, GLOBO H, GPA33, GPC3, GPNMB, HER2, p95HER2, HER3, HMW-MAA (high molecular weight melanoma associated antigen), integrin alpha (e.g. alphavbeta3 and alphavbeta5), IGF1R, TM4SF1 (L6), Lewis A-like glycochain, Lewis X, Lewis Y (CD174), LGR5, LIV1, mesothelin (MSLN), MN (CA9), MUC1, MUC16, NaPi2b, Nectin-4, Notch3, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, 5T4 (or TPBG, trophoblast glycoprotein), TF (tissue factor, thromboplastin, CD142), TF-Ag, Tag72, TNFα, TNFR, TROP2 (tumor associated calcium signal transducer 2), uPAR, VEGFR and VLA.

[0033] Examples of suitable antibodies known in the art include blinatumomab (CD19), rituximab (CD20) or other anti-CD20 antibodies such as ofatumumab, ublituximab or ocrelizumab, epratuzumab (CD22), iratumumab and brentuximab (CD30), gemtuzumab, vadastuximab (CD33), tetulumab (CD37), daratumumab, isatuximab (CD38), bivatuzumab (CD44), alemtuzumab (CD52), lorvotuzumab (CD56), borsetuzumab (CD70), milatuzumab ( CD74), polatuzumab (CD79), rovalpituzumab (DLL3), futuximab (EGFR), oportuzumab (EPCAM), farletuzumab (FOLR1), glembatumumab (GPNMB), trastuzumab, pertuzumab and margetuximab (HER2), etaracizumab (integrin), anetumab (mesothelin), pancomab (MUC1), enfortumab (nectin-4), H8, A1 and A3 (5T4), and antibodies against TROP2, such as sacituzumab, datopotamab and PF-06664178. Conjugates of the invention in which the targeting moiety is a tumor targeting antibody against CD20 (eg rituximab), HER2 (eg trastuzumab), or an anti-CD123 antibody are provided in the examples.

[0034] Since the pAg activity of the pAg moiety must be exerted intracellularly, an antibody that is internalized within cells is preferred.

[0035] The antibody or antigen-binding fragment thereof may, where applicable, comprise: (1) a constant region that may be engineered, i.e., one or more mutations may be introduced, for example, to increase half-life, to provide a site for linker-drug attachment, and / or to increase or decrease effector function; or (2) a variable region that may be engineered, i.e., one or more mutations may be introduced, for example, to provide a site for linker-drug attachment. The antibody or antigen-binding fragment thereof may be produced recombinantly, synthetically, or by other known suitable methods. Mutations that may decrease Fc-mediated effector function of the antibody are, for example, mutations such as those described in Leabman et al., 2013, MAbs, 5(6):896-903 and Bruhns P, et al., 2015, Immunol Rev., 268(1):25-51. doi: 10.1111 / imr.12350. PMID: 26497511.

[0036] Conjugates of the invention may be wild-type or site-directed (meaning that specific conjugation sites, e.g., cysteine ​​or unnatural amino acids, have been engineered into the antibody protein sequence), or a combination thereof, and can be produced by methods known in the art.

[0037] The immunoconjugates of the invention contain a phosphoantigen (pAg) as an immunomodulatory moiety. It has been found that the immunoconjugates of the invention deliver the pAg payload very efficiently to antigen-presenting cells, such as cancer cells, resulting in active phosphoantigens within the antigen-presenting cells. The antigen-presenting cells may be tumor cells that express or overexpress a particular tumor antigen on their surface. Such cells may also express or overexpress TCR activating molecules involved in the indirect activation of γδ T cells by pAg, such as BTN3A1 / BTN2A1 receptor complex molecules.

[0038] Phosphoantigen (pAg) Phosphoantigens include non-peptide antigens of relatively small mass that can stimulate γδ T cells (more specifically Vγ9Vδ2 cells) in the presence of antigen-presenting cells. Throughout this specification, the term "phosphoantigen" or "pAg" refers to naturally occurring phosphoantigens as well as non-naturally occurring (synthetic) pAgs or prodrugs thereof, including modified pAgs, e.g., analogs of naturally occurring pAgs. A pAg suitable for use in the present invention may be a pyrophosphate (diphosphate), pyrophosphonate, bisphosphonate (or diphosphonate), monophosphate or monophosphonate or a prodrug thereof. A preferred pAg for use in the conjugates and linker-drug compounds of the present invention is a (mono)phosphonate. A preferred phosphoantigen for use in the conjugates and linker-drug compounds of the present invention comprises an allyl alcohol group, e.g., an allyl alcohol group present in natural phosphoantigens, e.g., HMBPP. Preferably, the phosphoantigen is a monophosphonate comprising an allyl alcohol group.

[0039] The "phosphoantigen" as part of the conjugate or linker-drug compound of the present invention does not necessarily contain the phosphoantigen in its active form. The phosphoantigen in the conjugate or linker-drug compound may contain an inactive precursor form of the active phosphoantigen and / or may release the active phosphoantigen only after the conjugate binds to the target and is processed. Thus, the phosphoantigen in the bound state as part of the conjugate or linker-drug compound may be structurally different from the active phosphoantigen released therefrom. For example, separation from or cleavage of the linking moiety may initiate structural rearrangements and / or chemical or enzymatic reactions that lead to the formation of the functionally active phosphoantigen. Also, the prodrug moiety may be removed or rearranged, for example in response to a change in the environment or as a result of enzymatic activity at the target site, releasing the functionally active phosphoantigen.

[0040] It is believed that compounds with cellular pAg activity can be active directly by binding to a pAg receptor on target cells ("direct pAg"). This receptor is believed to be butyrophilin 3A1 (BTN3A1), an intracellular domain of a cell surface molecule. An example of a natural direct pAg is HMBPP. HMBPP is produced by pathogenic bacteria. It has been found that the allylic alcohol in natural pAgs such as HMBPP is important for maximizing BTN3A1 binding and pAg activity. Direct pAgs such as HMBPP bind to BTN3A1 directly at their intracellular B30.2 domain. Analogs of HMBPP, such as halohydrins such as BrHPP, IHPP, and ClHPP, are also known in the art (Wiemer et al., 2020, Chem. Med. Chem., 15, 1030-1039).

[0041] Other compounds exhibit indirect pAg activity by accumulating IPP. Such compounds can be referred to as "indirect pAgs". Indirect pAgs act on a pathway that increases the cellular levels of (endogenous) direct pAgs, such as IPP, and the coactivation of Vγ9Vδ2 T cells. In contrast to direct pAgs, indirect pAgs do not directly interact with butyrophilin receptors in target cells, nor are they pAg precursors (compounds that are enzymatically or chemically converted to direct pAgs). Indirect pAgs can be, for example, compounds that inhibit downstream enzymes, such as farnesyl pyrophosphate synthase (FPPS). Inhibition of FPPS prevents the use of IPP, resulting in the accumulation of IPP in cells. Aminobisphosphonates (N-BPs), such as zoledronate, are known as FPPS inhibitors (Wiemer et al., 2020, Chem. Med. Chem., 15, 1030-1039; Park et al., 2021, Frontiers in Chemistry, Vol. 8, Article 612728).

[0042] Aminobisphosphonates (N-BPs), such as zoledronate, pamidronate, and alendronate, are also known as “bone targeting agents” due to their ability to specifically bind to hydroxyapatite (HA) (Farrell et al., 2018, Bone Reports, 9, 47-60). Alendronate has also been conjugated to trastuzumab to target trastuzumab to bone metastases using its function as a bone targeting agent (Tian et al., 2021, Sci.Adv., 7, 2-11). Due to its negative charge, alendronate has a high affinity for HA and preferentially binds to bone. Therefore, Tian et al. proposed to use negatively charged aminobisphosphonates such as alendronate as targeting agents for antibodies to treat bone-related diseases.

[0043] In the conjugates of the invention, for example, specific binding between an antibody (the targeting moiety) and its specific binding partner (e.g., a tumor-specific antigen) directs the pAg to its target site, but not vice versa (the pAg is not the targeting moiety). In the conjugates of the invention, it is the binding specificity and affinity of the targeting moiety (e.g., an antibody) that ensures that the phosphoantigen is delivered to the site where it must exert its therapeutic effect.

[0044] Preferred pAgs for use in the present invention include allylic alcohols or prodrugs thereof (e.g., pAgs in which an allylic alcohol is generated after removal of the prodrug group or after cleavage of a linker moiety via or conjugated to the isoprene unit). Such compounds are considered to be examples of pAgs with direct pAg activity (pAgs that act as BTN3A1 ligands). Alternative precursors, for example compounds that are metabolized to compounds with (direct) pAg activity, or prodrugs or precursors of direct pAgs, can be used as pAgs in the conjugates of the present invention.

[0045] The activity of phosphoantigens on Vy9V52 T cells can be measured in a cell assay, as illustrated in the Examples. In the cell-based assay used, in a first step target cells, e.g. tumor cells, e.g. cells from the CD20 positive Burkitt's lymphoma human tumor cell line Raji, are incubated (overnight) with the phosphoantigen or a phosphoantigen-containing complex of the invention.

[0046] In this first step, the phosphoantigen or the complex of the invention is taken up into the target (tumor) cell. After internalization (and in the case of the complex, cleavage of the linker), the phosphoantigen binds to the intracellular domain of the BTN3A1 receptor, which results in the activation of the BTN3A1 / BTN2A1 dimer.

[0047] The pretreated and washed tumor cells from the first step can be co-cultured with γδ T cells in the second step. When Vγ9Vδ2 T cells are activated, the cells produce cytokines to activate the immune system and release cytotoxic granules (degranulation) to kill target cells.

[0048] To assess the activity of phosphoantigens in γδ T cells, monensin and / or brefeldin A are added to co-cultures of γδ T cells and targets. This captures cytokines produced by activated cells (e.g., interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα)). Staining with fluorescently labeled antibodies in the presence of saponin, which allows the anti-cytokine antibodies to be internalized, identifies cytokine-producing cells. Fluorescently labeled antibodies against CD107a can also be added to the co-cultures to stain cells undergoing degranulation. Degranulation correlates with tumor cell killing (Aktas et al., 2009, Cell Immunol., 254(2),149-154).

[0049] Thus, by combining fluorescently labeled immune cell specific markers with CD107a and cytokine markers, it is possible to determine the activation state of γδ T cells and / or other immune cell subsets after co-culture with pretreated target cells.

[0050] The ability of γδ T cells to kill pre-treated tumor cells can be examined by determining the percentage of dead tumor cells after co-culture. Tumor cells can be easily identified by fluorescent tags, and their dead cells can be determined as early as 1 h after co-culture with γδ T cells.

[0051] Phosphoantigen analogs A (chemical) analogue is a compound that differs from the native phosphoantigen in structural features but is similar to the native phosphoantigen in functional biological activity, particularly exhibiting (indirect) immunostimulatory activity on γδ T cells. Analogues can be designed to improve one or more characteristics of the naturally occurring pAg, such as stability, potency, bioavailability or binding to a linking moiety, in the context of their use in the immunoconjugates and linker-drug compounds of the invention. Native phosphoantigens include pyrophosphates (diphosphates), such as HMBPP and IPP. Known analogues of native phosphoantigens include bromohydrin pyrophosphate (BrHPP) and pyrophosphonates, such as C-HMBPP. Phosphonates with phosphoantigen activity known in the art further include bisphosphonates that differ in the substitution of the central carbon between the two phosphate groups. Examples include etidronate, clodronate, tiludronate, and a group of bisphosphonates that have a nitrogen or amino group in one of the central carbon substituents that is believed to increase the potency of the bisphosphonate (Drake et al., Mayo Clin. Proc., 2008, 83(9), 1032-1045). Phosphoantigens of these nitrogen-containing bisphosphonates include zoledronate (zoledronic acid), alendronate, risedronate, ibandronate, pamidronate, neridronate, and olpadronate.

[0052] Another phosphoantigen analogue, a phosphoramidite ester, with allegedly increased potency is described in WO 2005 / 05258 (Innate Pharma), e.g., N-HDMAPP, in which the isoprene unit present in natural HMBPP is linked to a pyrophosphate via its NH group.

[0053] Phosphoantigen Prodrugs For prodrugs, an inactive precursor of a phosphoantigen is meant one which is converted to an active phosphoantigen after removal or conversion of a protecting group (e.g. a neutral protecting group on the negatively charged non-bonding oxygen atom of a phosphonate group). After a conjugate comprising a phosphoantigen of the invention in the form of a prodrug is administered to the body, the protecting group may be metabolically removed at the target site. A prodrug may be formed by binding of a phosphoantigen to a linking moiety. In this case, an active phosphoantigen may be formed because a linker of the conjugate used to connect the phosphoantigen prodrug moiety to the targeting moiety is cleaved to release the active phosphoantigen and / or because a protecting group is removed from the phosphoantigen. Preferably, such conversion releasing the active phosphoantigen only occurs after the conjugate of the invention has reached the site where it must exert its therapeutic effect, e.g. after it has been internalized into tumor cells, or at least within the tumor microenvironment, in order to prevent unwanted and non-specific side effects of the phosphoantigen in healthy and / or non-target tissues.

[0054] For example, certain bisphosphonates have a high affinity for bone mineral and are used as "bone targeting agents." Such bisphosphonates act as targeting molecules for different drugs conjugated to them, targeting the drugs to bone, where the drugs exert their therapeutic effect, for example, on cells localized in bone (Farrell et al., 2018, Bone Reports, 9, 47-60).

[0055] In contrast, in the conjugates of the invention, the phosphoantigen is conjugated to a targeting moiety (e.g., a tumor-specific antibody). In the conjugates of the invention, it is the binding specificity of the targeting moiety that ensures that the phosphoantigen is delivered to the site where it must exert its therapeutic effect.

[0056] In the present invention, unwanted reactivity of phosphoantigens (e.g., binding of the phosphoantigen itself to non-target tissues) can be further prevented by including a prodrug of the phosphoantigen in the conjugate rather than the active phosphoantigen.

[0057] For example, the negatively charged phosphonate group of a bisphosphonate may be masked with a prodrug moiety that is delivered to the target site by the targeting moiety of the conjugate where it is converted to the active phosphoantigen.

[0058] Prodrug types include protecting groups known in the art, such as aryl ester, aryl amide, or pivaloyloxymethyl (POM) prodrugs. C-HMBP (monophosphonate) phosphoantigen analogs / prodrugs are described in WO 2019 / 182904. With the aim of synthesizing phosphoantigen prodrugs that are as potent as natural phosphoantigens, such as HMBPP, aryloxytriester phosphoramidite prodrugs of (monophosphonate) phosphoantigens were synthesized as described in Davey et al., 2018, J. Med. Chem., 61, 2111-2117. In these prodrugs, the monophosphonate group is masked by an aryl motif and an amino acid ester moiety. These compounds ("HMBP ProPagens") were rather poorly stable in serum due to cleavage of the -PO- bond between the phosphate and isoprenoid moieties in the molecule. Similar "ProPagens" compounds, in which the oxygen in the -PO- bond is replaced by carbon, are described in WO 2020 / 008189. A proposed structure-activity relationship (SAR) for phosphoantigens (prodrugs) is described by Wiemer et al., 2020, Chem.Med.Chem., 15, 1030-1039.

[0059] The cleavable linking moiety may be covalently attached to the phosphoantigen via the alcohol group of an allylic alcohol, in which case the allylic alcohol is (re)formed intracellularly when the cleavable linking moiety is cleaved.

[0060] The prodrug moieties in a phosphoantigen prodrug as part of a conjugate of the invention may be the same or different, for example all the prodrug moieties may be POM groups or the phosphoantigen may include, for example, a "proTide" group, such as a combination of aryloxy and amino acid ester radicals, such as those described for phosphoantigen prodrugs in WO2020 / 008189 or WO2019 / 182904.

[0061] Suitable phosphonate prodrug technology and the synthesis of phosphonate prodrugs are known in the art. Such prodrug technologies are further reviewed, for example, in Pradere et al., 2014, Chem. Rev., 114, 9154-9218, and include the use of carbonyloxymethyl prodrug moieties, such as pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) derivatives, S-acyl-2-thioethyl (SATE) and S-[(2-hydroxyethyl)sulfidyl]-2-thioethyl (DTE)-based prodrugs, cyclosaligenyl (cycloSal) phosphate and phosphonate-based prodrugs and alkoxyalkyl monoester (hexadecyloxypropyl-(HDP), octadecyloxyethyl-(ODE))-based prodrugs, phosphoramidite and phosphonamidite-based prodrugs (including aryloxyamino acid amidate (ProTide) prodrugs), and phosphorodiamidates and phosphonodiamidates.

[0062] Linker-drug compound The present invention also provides linker-drug compounds comprising at least one phosphoantigen covalently bound to a linking moiety. Such linker-drug compounds may be used as intermediates in the synthesis of the conjugates of the present invention. For example, when the targeting moiety is an antibody or an antigen-binding fragment thereof, one or more linker-drug compounds of the present invention can be conjugated to the targeting antibody to produce the conjugates of the present invention.

[0063] The linker-drug compounds of the invention comprise at least one phosphoantigen (pAg or "drug") and a linking moiety (L or "linker"). Such linker-drug molecules can be used to prepare the conjugates of the invention. Preferred linker-drug compounds of the invention have the general formula II: [ka] (In the formula, Q is a group represented by formula IIa or IIb: [ka] The structure is shown in Y is a halogen; W 1 is N, CH or CF, preferably CH; W 2 is CH2, CHF, CF2 or O; X 1 is O, S, NH, CH2, CHF or CF2; X 2 is O, CH2, CHF or CF2; X 3 is absent or is O or NH; X 4a~d are each independently selected from O and S; X 5 is CH3, CH2F, CHF2, CF3 or CCl3; x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R 1 is H or a connection to a linking moiety (L) or a prodrug moiety; R 2 is H or a connection to a linking moiety (L), Cat+ or a prodrug moiety; R 3 is H or a connection to a linking moiety (L), Cat+ or a prodrug moiety; R 4 is H or a connection to a linking moiety (L), Cat+ or a prodrug moiety; Alternatively, if n is 0, then R 3 and R 2 is C 1~6 are connected by a (hetero)alkyl group; or When n is 1 or 2, R 3 and R 4 is C 1~6 (Hetero)alkyl groups It may be represented as:

[0064] Linker-drug compounds in which Q has the structure of formula IIb may have a phosphoantigen similar to an (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) analog, such as BrHPP (phosphostim), IHPP, or ClHPP.

[0065] In a preferred embodiment of the present invention, the linker-drug compound includes a compound of formula II, where Q is a structure of formula IIa. Such compounds have the general formula III: [ka] (In the formula, W 1 is N, CH or CF, preferably CH; W 2 is CH2, CHF, CF2 or O; X 1 is O, S, NH, CH2, CHF or CF2; X 2 is O, CH2, CHF or CF2; X 3 is absent or is O or NH; X 4a~d are each independently selected from O and S; X 5 is CH3, CH2F, CHF2, CF3 or CCl3; x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R 1 is H or a connection to a linking moiety (L) or a prodrug moiety; R 2 is H or a connection to a linking moiety (L), Cat+ or a prodrug moiety; R 3 is H or a connection to a linking moiety (L), Cat+ or a prodrug moiety; R 4is H or a connection to a linking moiety (L), Cat+ or a prodrug moiety; Alternatively, if n is 0, then R 3 and R 2 is C 1~6 are connected by a (hetero)alkyl group; or When n is 1 or 2, R 3 and R 4 is C 1~6 (Hetero)alkyl groups It is expressed as:

[0066] The structural formula between the outer square brackets represents the pAg, and L represents the linker moiety. There is only one connection to the linking moiety per linker-drug molecule (however, if x is greater than 1, then multiple pAgs are attached to one (branched) linker moiety).

[0067] Preferably, n is 0 or 1, most preferably 0. When n is 1 or 2, X 2 is preferably O. n is 1 and X 2 is CH2 or n is 1 and X 2 Linker-drug compounds having a phosphoantigen of O are also part of the invention. In this case, each X 4a~d is preferably O. In such linker-drug compounds, R 3 or R 1 may be a connector with a linking moiety, preferably R 3 represents the connection part with the connecting part.

[0068] If n is 2, then X 2 occurs twice in formula II, and X 2a and X 2b which may be independently selected from O, CH2, CHF, and CF2. When n is 2, R 4 Also appears twice, R 4a and R 4b and can be independently selected from H, the connection to the linking moiety (L), the connection to Cat+, and the connection to the prodrug moiety. 4cand X 4d The same is true for X 4c , X 4ci , X 4d and X 4di ) may occur and be independently selected from O and S.

[0069] When m is 2 or 3, W 2 occurs multiple times in formula II, and each W 2 can be independently selected from CH2, CHF, CF2 or O. Preferably, W 2 is CH2. In a preferred embodiment, m is 1, and most preferably, when m is 1, W 2 is CH2.

[0070] Cat+ represents a positive ion (organic or inorganic), including a hydrogen ion.

[0071] X 1 is preferably CH2, O or S, most preferably CH2.

[0072] each X 4a~d (when present) is preferably O. n is 1 or 0, and X 4a~b and X 4c~d (if present) is O and R 2 and R 4 Compounds where (when present) is preferably H are part of the invention. Preferably, n is 0 and X 4a and X 4b is O and R 2 is preferably H.

[0073] In the linker-drug compound in which Q has the structure shown in formula IIa, W 1 is preferably CH or CF, most preferably CH.

[0074] When Q is the structure of formula IIa, X 5 is preferably CH3.

[0075] In the linker-drug compound in which Q has the structure shown in formula IIa, R1 is preferably H or a junction with the linking moiety (L), most preferably H.

[0076] In linker-drug compounds in which Q has the structure of formula IIa, preferably W 1 is CH, and X 5 is CH3, X 1 is CH2 and R1 is H, resulting in a linker-drug molecule having a pAg with an allylic alcohol group. 1 is CH and X 5 is CH3, X 1 When R1 is H, W2 is CH2 and m is 1. 1 is CH and W 2 is CH2, m is 1, and X 5 is CH3, X 1 When R is O and R is H, the phosphoantigen comprises an aryl alcohol chain present in a natural phosphoantigen, such as HMBPP.

[0077] Preferred linker-drug compounds are those in which Q has the structure of formula IIa: 3 is O and R 3 At the connection with the disconnectable connecting part, W 1 is CH and X 5 is CH3, R 1 is H and W 2 is CH2, m is 1, and X 1 is that of CH2.

[0078] In such compounds, R 2 , R 3 and / or R 4 may be a prodrug moiety alone, or X 4b , X 4d and / or X 3 (if present) may be a prodrug moiety in combination with -X 4b -R 2 , -X 4d -R 4 and / or -X 3 -R 3 (It is.)

[0079] Preferably, in the linker-drug compound in which Q has the structure of formula IIa, W 1 is CH, W 2 is CH2, X 4a~d is O and R 2 and R 4 is H, and X 5 is CH3 and m is 1.

[0080] In a preferred embodiment, Q is the structure shown in formula IIa, W 1 is CH, W 2 is CH2, n is 0, and X 4a~b is O and X 5 is CH3 and m is 1.

[0081] In formula II, x represents the number of phosphoantigens (pAg) per linking moiety (L), and the structure between the brackets is preferably the structure of a phosphoantigen for use in the linker-drug compounds of the invention. X can be an integer from 1 to 5 (each linking moiety has 1 to 5 pAgs). Preferably, the linking moiety has 1 or 2 pAgs. In most cases, one pAg per linking moiety will be sufficient.

[0082] The connection part with the connecting part is R 1 Alternatively, the linking moiety may be (part of) R 2 , R 3 Or R 4 may be (may be connected with). Preferably, R 1 or R 3 , more preferably R 3 is the connection part with the connecting part. The connecting part is R 3 If connected via X 3 is preferably O. 3 When X is the connection to the linker moiety, it is preferable 3 is O and R 1 is preferably H.

[0083] The connecting part is R 2 or R 4 If they are joined by X 4b Or X4d is preferably O.

[0084] Preferred linker-drug compounds are those in which Q has the structure of formula IIa: 3 is O and R 3 is the connection with the cleavable linking moiety, preferably W 1 is CH and X 5 is CH3, R 1 is H and W 2 is CH2, m is 1, and X 1 is CH2. In such compounds, n is preferably 0.

[0085] "Connection to the linking moiety" refers to the position in the molecule where the linker is attached to the phosphoantigen. 1 , R 2 , R 3 or R 4 (depending on where the linker is attached) is not necessarily meant to represent the actual (remaining) structural element of the linker-drug compound between the linker and the remainder of the phosphoantigen. For example, depending on the linker used, R 1 When R represents the connection with the linking moiety, this also includes the situation where the linker is directly bonded to an oxygen atom of the phosphoantigen in the linker-drug molecule. 1 is the connection part with the connecting part (L). R 1 When W is a connecting portion with the connecting portion (L), W 1 is CH, W 2 is CH2, m is 1, and X 5 is CH3, X 1 When such a linker-drug molecule is incorporated into a conjugate of the invention, cleavage of the linker after administration results in the release of an aryl alcohol group (R 1 H) may be (re)formed. 1may also be a prodrug moiety. Suitable alcohol prodrug moieties are known in the art. For example, an alcohol can be masked by an ester-based prodrug group. The generation of an active alcohol relies on hydrolysis of the ester bond by (cellular) esterases, which metabolically regenerates the alcohol (drug) and the carboxylic acid (free radical).

[0086] R 2 , R 3 and R 4 may each independently be H or a connection to a linking moiety (L), Cat+ or a prodrug moiety. In a preferred embodiment, the compounds of the invention are monophosphonates (n is 0), and thus R 4 does not exist.

[0087] Cat+ represents a cation (organic or inorganic) including hydrogen ion (and may be exchanged in formulation buffer or plasma). 2 , R 3 and / or R 4 When R is Cat+, Cat+ may be the same or different. 2 , R 3 and / or R 4 If is Cat+, then X 4b and X 4d (present, i.e., if n is not 0) and / or X 3 is O, and O - Cat + This results in:

[0088] In another embodiment of the invention, when n is 0, R 3 and R 2 is C 1~6 are connected by a (hetero)alkyl group. In this case, R 3 and R 2 together form a substituted or unsubstituted 5-8 membered ring. In such embodiments, preferably the linking moiety is R 1 In another embodiment, when n is not 0, R 3 and R 4 is C1~6 They may also be attached in a similar manner via (hetero)alkyl groups.

[0089] R 2 , R 3 and / or R 4 may also be a prodrug moiety alone, or X 4b , X 4d and / or X 3 (if present) may be a prodrug moiety in combination with -X 4b -R 2 , -X 4d -R 4 and / or -X 3 -R 3 ).

[0090] A "prodrug moiety" may be a group that is cleaved (releasing an active compound) non-enzymatically or enzymatically. A "prodrug moiety" may induce the release of a second prodrug moiety at another location in the molecule after the conjugate of the invention is administered to a subject. Preferably, the phosphoantigen in the form of a prodrug is converted to a functionally active phosphoantigen inside a target cell (e.g., a tumor cell), e.g., by enzymatic removal of the prodrug moiety.

[0091] Examples of prodrug technology known in the art include the use of pivaloyloxymethyl (POM) or isopropyloxycarbonyloxymethyl (POC) groups. In a preferred embodiment, at least R 2 and R 3 is independently selected from a POM group or a POC group (e.g., when n is 0). When n is 1 or 2, R 4 may likewise be a POM or POC group.

[0092] Phosphoantigen prodrugs of this type are described, for example, in WO 2019 / 182904. Such phosphoantigen prodrugs can be used as pAg sources in the linker-drug compounds and conjugates of the invention.

[0093] In another example, a combination of free radicals can be used. An example of such a prodrug technology is the "ProTide" technology, which has been developed for intracellular delivery of monophosphates and monophosphonates. The hydroxyl of the monophosphate or monophosphonate group in the ProTide prodrug is masked (or replaced) by an aromatic group and an amino acid ester moiety, which are enzymatically cleaved in the cell to release the free monophosphate and monophosphonate (Mehellou et al. 2018, Journal of Medicinal Chemistry, 61(6), 2211-2226).

[0094] Thus, if the phosphoantigen is a monophosphate or monophosphonate, and R 2 and R 3 Linker-drug compounds and conjugates of the invention in which R is a combination of a "ProTide" radical are also part of the invention. 2 is an aromatic moiety and R 3 is an amino acid ester moiety, or vice versa. In a preferred embodiment of the present invention, when n is 0, R 2 or R 3 may be a substituted or unsubstituted (hetero)aryl group, R 2 or R 3 The other is represented by formula IV and formula V: [ka] (In formula; R a and R a’ represents H, an optionally substituted amino acid side chain, and an optionally substituted C 1~14 independently selected from non-polar side chains comprising alkyl chains; R b is H, benzyl or substituted or unsubstituted (C1-8)-alkyl; R c and R c’is independently selected from H, or optionally substituted (C1-6)-alkyl, (C3-6)cycloalkyl, aryl or heteroaryl, or R c and R c’ may, together with the nitrogen to which they are attached, form an optionally substituted ring, for example, an aziridino, azetidino, morpholino, piperazino, pyrrolidino or piperidino ring. The structure may be selected from those shown in

[0095] R c and / or R c’ The substituents in are bioisosteres of carboxylic acids, amino, tetrazole, sulfonate, hydroxyl, halo, or alkyl.

[0096] R b When is a substituted alkyl, the substituents are hydroxy, amino, halo, nitro, cyano, carboxy, NR x R y , (C1-6)alkoxy, (C1-6)alkanoyl, (C1-6)alkoxycarbonyl, (C1-6)alkylthio, and (C2-6)alkanoyloxy; R x and R y is independently selected from the group consisting of H, (C1-C6)alkyl, (C3-6)cycloalkyl, and (C3-6)cycloalkyl(C1-6)alkyl; or R x and R y together with the nitrogen to which they are attached form an aziridino, azetidino, morpholino, piperazino, pyrrolidino, or piperidino group.

[0097] connecting part The linking moiety (or "linker") used in the conjugates or linker-drug compounds of the invention is preferably a synthetic linker. The structure of the linker is such that it can be readily chemically coupled to a low molecular weight effector molecule (phosphoantigen) and the resulting linker-drug compound can be readily conjugated to another agent, such as a polypeptide (e.g., an antibody). The choice of linker can affect the stability of the conjugate in blood and, if any, the manner in which the low molecular weight effector compound (phosphoantigen) is released. Suitable linkers are described, for example, in Ducry et al, 2010, Bioconjugate Chem., 21, 5-13;King and Wagner, 2014, Bioconjugate Chem., 25, 825-839;Gordon et al., 2015, Bioconjugate Chem., 26, 2198-2215;Tsuchikama and An, 2018, Protein & Cell, 9, 33-46 DOI: 10.1007 / s13238-016-0323-0;Polakis, 2016, Pharmacological Reviews, 68 (1), 3-19, DOI: 10.1124 / pr.114.009373;Bargh et al., 2019, Chem. Soc. Rev., 48, 4361-4374, DOI: 10.1039 / c8cs00676h, WO 02 / 83180, WO 2004 / 043493, WO 2010 / 062171, WO 2011 / 133039, WO 2015 / 177360 and WO 2018 / 069375. The linker may be cleavable or non-cleavable, for example, as described in van Delft, F and Lambert, JM, 2021, Chemical Linkers in Antibody-Drug Conjugates (ADCs), 1st Ed. Royal Society of Chemistry, ISBN-10: 1839162635.Another method of conjugating a linker-drug to an antibody is to use a transpeptidase, such as bacterial sortase or plant asparaginyl endopeptidase, to allow site-specific conjugation of a chemical moiety linked to an appropriate synthetic peptide. Sortase A (Sort-A) recognizes a C-terminal peptide sequence (LPXTG) and creates a bond between a threonine in this sequence and a glycine at the N-terminus of the conjugation partner (e.g., an ADC that is a glycine-tagged payload) (Combs et al., 2015, the AAPS Journal, Vol. 17, No. 2, 339-351, DOI: 10.1208 / s12248-014-9710-8). Antibody-drug conjugation can also be achieved by site-specific glycoengineering, for example, using endo-β-N-acetylglucosaminidase (ENGase) and monosaccharyltransferase mutants (Manabe et al., 2021, Chem Rec, (11),3005-3014, doi: 10.1002 / tcr.202100054; Wang et al., 2019, Annu Rev Biochem, 20;88,433-459, doi: 10.1146 / annurev-biochem-062917-012911).

[0098] The use of cleavable linkers in the conjugates of the invention is preferred. Cleavable linkers include moieties that are cleaved, for example, when exposed to lysosomal proteases or to an environment of acidic pH or high reductive potential. Suitable cleavable linkers are known in the art and include, for example, mono-, di-, tri- or tetrapeptides, i.e., 1, 2, 3 or 4 amino acid residues. Additionally, cleavable linkers may include self-immolative moieties, for example, ω-amino aminocarbonyl cyclization spacers (see Saari et al, 1990, J. Med. Chem., 33, 97-101) or -NH-CH2-O-. Cleavage of the linker renders the immunomodulatory effector moiety (phosphoantigen or "pAg" moiety) in the conjugates of the invention available to its surrounding environment. Non-cleavable linkers can also effectively release (active derivatives of) phosphoantigens from the immune conjugates of the invention, for example, when the conjugated polypeptide (antibody) is degraded in the lysosome. Non-cleavable linkers include, for example, succinimidyl-4-(N-maleimidomethyl(cyclohexane)-1-carboxylate and maleimidocaproic acid and analogs thereof.

[0099] To allow the conjugation of a linking moiety or linker-drug compound to a polypeptide such as an antibody, the linking moiety that is (covalently) bound to the antibody usually has a functional group that can react with an amino acid residue of the antibody under relatively mild conditions. This functional group is referred to herein as a reactive moiety (RM). Examples of reactive moieties include, but are not limited to, carbamoyl halides, acyl halides, active esters, anhydrides, α-haloacetyl, α-haloacetamide, maleimide, isocyanate, isothiocyanate, disulfide, thiol, hydrazine, hydrazide, sulfonyl chloride, aldehyde, methyl ketone, vinyl sulfone, halomethyl, methyl sulfonate, cyclooctyne, and trans-cyclooctene (TCO). The amino acid residue that reacts with the functional group may be a natural or non-natural amino acid residue, or a (non-)natural glycan (Manabe et al., Wang et al., supra). In this specification, the term "unnatural amino acid" is intended to denote a (synthetically) modified amino acid or a D-stereoisomer of a naturally occurring amino acid. Preferably, the amino acid residue with which the functional group is reacted is a natural amino acid.

[0100] The linking moiety (L) used in the conjugate or linker-drug compounds of the invention has Formula VI or Formula VII: [ka] (wherein m is an integer of 1 to 10, preferably 5; A is an amino acid, preferably a natural amino acid; and p is 0, 1, 2, 3, or 4. When p is greater than 1, the amino acids may be the same or different.) It may include a structure shown in

[0101] Suitable amino acid combinations are known in the art and include amino acids selected from the group consisting of alanine, glycine, lysine, phenylalanine, valine and citrulline. Preferably, p is 2. When p is 2, AA2 may be, for example, phenylalanyl lysine, valyl alanine, valyl citrulline or valyl lysine. When p is 2, AA2 is preferably valyl alanine or valyl citrulline. When p is 3, AA3 may be, for example, alanyl phenylalanyl lysine, and when p is 4, AA4 may be, for example, glycyl glycyl phenylalanyl glycine.

[0102] "q" is an integer from 1 to 12, preferably 2; ES is absent or an extended spacer selected from the following: [ka] (In the formula, R 5 is H, halogen, CF3, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxyl or C1-4 alkylthio, preferably H, F, CH3 or CF3, more preferably H or F; V is H, ethyl, -(CH2CH2O) p -OMe, CH2CH2SO2Me, or CH2CH2N(Me)2, and p is an integer from 1 to 12.

[0103] Linking moieties may be branched, resulting in a single linking moiety that can carry multiple phosphoantigens. Examples of branched linking moieties are: [ka]

[0104] These branched linker moieties can be used to obtain conjugates with relatively high pAg to targeting moiety ratios ("DAR"). Such branched linkers can be used to synthesize conjugates with DARs of 16 and even 20 or more. The DAR of antibody-based conjugates of the invention may be as low as about 2. However, for antibodies against tumor-specific targets known to be relatively poorly expressed in target tumor cells, conjugates with high pAg to targeting moiety ratios may be preferred. Linker-drug compounds for use in the linker-drug compounds of the invention may contain, for example, a linking moiety selected from the following:

[0105] [ka]

[0106] [ka]

[0107] The linker moiety (L) may be attached to the pAg to give a linker-drug compound of the general formula shown in Formula II.

[0108] The linker-drug compounds of the invention can be conjugated to a targeting moiety to produce a conjugate of the invention. A preferred conjugate of the invention comprises a tumor-targeting antibody or antigen-binding fragment thereof conjugated to a linker-drug compound of the invention.

[0109] In a specific embodiment of the invention, the phosphoantigen as part of the conjugate of the invention is a monophosphonate prodrug, in which the negatively charged non-bonded oxygen atom of the phosphonate group is protected by a combination of ProTide moieties (a (hetero)aryl group and an amino ester radical) or one or more prodrug moieties such as POM or POC, while the cleavable linking moiety may be attached to an isoprene unit in the phosphoantigen molecule that is converted to an allylic alcohol present in the phosphoantigen, such as HMBPP, upon cleavage of the linker.

[0110] It should be understood that a linker-drug compound comprising at least one phosphoantigen covalently bonded to a linking moiety of the invention, when included in a conjugate of the invention, may lose or gain a certain atom or group of atoms, e.g., it may lose a hydrogen atom, compared to the same linker-drug compound of the invention when not included in a conjugate. This may be, for example, because the linker-drug compound of the invention is conjugated to a polypeptide via, for example, esterification to a hydroxyl moiety.

[0111] Examples of the synthesis of linker-drug molecules of the invention are further illustrated in the Examples. An example of a preferred linker-drug compound of the invention is XD18, of the following structural formula: [ka]

[0112] Linker-drug XD18-based conjugates of the invention may contain 1-20, preferably 1-8, more preferably 2 linker-drug molecules per antibody (e.g., rituximab, as illustrated in the Examples).

[0113] Synthesis of the Conjugates of the Invention To synthesize the conjugates of the present invention, one or more linker-drug compounds of the present invention may be coupled to a suitable targeting moiety. When the targeting moiety is a polypeptide (antibody or binding fragment thereof), the linker-drug compound may be conjugated via a reactive natural amino acid residue present in the suitable polypeptide, such as lysine or cysteine, or via the N-terminus or C-terminus. Alternatively, natural or non-natural reactive amino acid residues may be engineered into the suitable polypeptide, or reactive groups may be introduced by post-translational modification.

[0114] Conjugates of the invention may be prepared by conjugating a linker-drug compound of the invention to an antibody or antigen-binding fragment thereof, e.g., via a lysine ε-amino group of the antibody, preferably using an intermediate containing an amine-reactive group, e.g., an activated ester. Such methods are known for preparing conventional antibody-drug conjugates (ADCs).

[0115] Alternatively, immunoconjugates can be prepared by conjugating linkers via the free thiol of the side chain of cysteine ​​generated by reduction of the interchain disulfide bond using methods and conditions known in the art, see, e.g., Doronina et al, 2006, Bioconjugate Chem., 17, 114-124. The preparation process involves partial reduction of the solvent-exposed interchain disulfide, followed by modification of the resulting thiol with a Michael acceptor-containing linker, e.g., maleimide-containing linker, alpha-haloacetamide or ester. The cysteine ​​conjugation strategy results in up to two linkers-containing linker-drugs per reduced disulfide.

[0116] The preferred antibody used as a targeting moiety in the conjugates of the present invention is human IgG. Most human IgG molecules have four solvent-exposed disulfide bonds, which equates to an integer of 0 to 8 linked moieties per antibody. The exact number of phosphoantigens bound per targeting moiety depends on the number of phosphoantigens per linked moiety, the degree of disulfide reduction, and the molar equivalents of linker-drug containing linkers in the subsequent conjugation reaction. Complete reduction of all four disulfide bonds gives a homogenous construct with 8 linker moieties per antibody, while partial reduction usually results in a heterogeneous mixture with 0, 2, 4, 6 or 8 linked moieties per antibody.

[0117] In a preferred embodiment, the present invention relates to a conjugate in which a linker-drug compound of the present invention is attached to an antibody or antigen-binding fragment thereof via a cysteine ​​residue of the antibody or antigen-binding fragment thereof.

[0118] Site-specific conjugation with an antibody or antigen-binding fragment thereof Because antibodies contain many lysine residues and cysteine ​​disulfide bonds, conventional conjugation results in heterogeneous mixtures that present challenges for analytical characterization and manufacturing. Moreover, the individual components of these mixtures exhibit different physicochemical properties and pharmacology with respect to their pharmacokinetics, efficacy and safety profiles, hindering a rational approach to optimizing this modality.

[0119] To improve the homogeneity of the conjugates, the antibodies used in the (immuno)conjugates of the invention may be modified to allow site-specific conjugation of linkers. Methods for site-specific conjugation of drugs to antibodies are comprehensively reviewed in CR Behrens and B. Liu, 2014, mAbs, 6 (1), 1-8, and described in WO 2015 / 177360, WO 2005 / 084390 and WO 2006 / 034488.

[0120] Site-specific immunoconjugates are preferably prepared by conjugating a linker-drug compound to an antibody or antigen-binding fragment thereof through the side chain of an engineered cysteine ​​residue at an appropriate position of the mutant antibody or antigen-binding fragment thereof. The engineered cysteine ​​is usually capped with another thiol, e.g., cysteine ​​or glutathione, to form a disulfide. These capped residues must be uncapped before linker-drug conjugation can be performed. Conjugation of the linker-drug to the engineered residue can be achieved by (1) reducing both the native interchain disulfide and the mutant disulfide, then reoxidizing the native interchain cysteine ​​using a mild oxidizing agent such as CuSO4 or dehydroascorbic acid, followed by standard conjugation of the uncapped engineered cysteine ​​to the linker-drug, or (2) using a mild reducing agent that reduces the mutant disulfide faster than the interchain disulfide bond, followed by standard conjugation of the uncapped engineered cysteine ​​to the linker-drug. Suitable methods for site-specific conjugation of linker-drugs can be found, for example, in WO 2015 / 177360, which describes reduction and reoxidation, WO 2017 / 137628, which describes a method using mild reducing agents, and WO 2018 / 215427, which describes a method for coupling reduced interchain cysteines with uncapped engineered cysteines.

[0121] Pharmaceutical Compositions In another aspect, the present invention relates to a composition comprising the complex of the present invention, preferably the composition is a pharmaceutical composition, more preferably further comprising one or more pharma- ceutically acceptable additives. Such a composition is hereinafter referred to as the composition of the present invention. The composition may be a liquid formulation, a lyophilized formulation, or may be in the form of a capsule or tablet.

[0122] A pharmaceutical composition comprising the immunoconjugate of the invention is usually in the form of a lyophilized cake (lyophilized powder) which requires dissolution (i.e. reconstitution) (in water) before intravenous injection, or a frozen (aqueous) solution which requires thawing before use. Thus, in a preferred embodiment, the invention relates to a lyophilized composition comprising the immunoconjugate of the invention, preferably the composition is a pharmaceutical composition, more preferably it further comprises one or more pharma- ceutically acceptable additives. In another preferred embodiment, the invention relates to a frozen composition comprising water and the immunoconjugate of the invention, preferably the composition is a pharmaceutical composition, more preferably it further comprises one or more pharma- ceutically acceptable additives. In this context, the frozen solution is preferably at atmospheric pressure and is preferably obtained by freezing a liquid composition of the invention below 0°C. Suitable pharma- ceutically acceptable additives to be incorporated into the pharmaceutical composition of the present invention (before lyophilization) include buffers (e.g., aqueous solutions of amino acids such as citric acid, histidine, or salts with succinic acid), lyoprotectants (e.g., sucrose, trehalose), osmolality regulators (e.g., chloride salts such as sodium chloride), surfactants (e.g., polysorbates), and bulking agents (e.g., mannitol, glycine). Additives used in lyophilized protein formulations are selected based on their ability to prevent protein denaturation during the lyophilization process and storage.

[0123] medical use In another aspect, the present invention provides a conjugate of the invention or a composition of the invention for use as a medicament, preferably for treating cancer, an autoimmune disease or an infectious disease. The conjugate of the invention can for example be used to induce γδ T cell cytotoxicity against tumor cells and / or infected cells.

[0124] The complexes and compositions are hereinafter collectively referred to as articles of manufacture for use in the present invention.

[0125] In a first embodiment, the product of the invention finds use in the treatment of solid tumors or hematological malignancies. In a second embodiment, the product of the invention finds use in the treatment of autoimmune diseases. In a third embodiment, the product of the invention finds use in the treatment of infectious diseases, such as bacterial, viral, fungal, parasitic or other infections.

[0126] The cancer according to the present invention is preferably a tumor expressing the antigen to which the product used in the present invention binds. Such a tumor may be a solid tumor or a hematological malignancy. Examples of tumors or hematological malignancies treatable with the product used in the present invention as described above include breast cancer; brain cancer (e.g. glioblastoma); head and neck cancer; thyroid cancer; parotid cancer, adrenal cancer (e.g. neuroblastoma, paraganglioma or pheochromocytoma); bone cancer (e.g. osteosarcoma); soft tissue sarcoma (STS); eye cancer (e.g. uveal melanoma); esophageal cancer; gastric cancer; small intestine cancer; colorectal cancer; urothelial cell cancer (e.g. bladder, penile, ureter or kidney cancer); ovarian cancer; uterine cancer; vaginal cancer, vulvar cancer and cervical cancer; lung cancer (particularly non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)); melanoma; mesothelioma (particularly malignant pleural and peritoneal mesothelioma); liver. These include, but are not limited to, cancer (e.g., hepatocellular carcinoma); pancreatic cancer; skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma, or dermatofibrosarcoma protuberans); testicular cancer; prostate cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); myelodysplastic syndromes (MDS); blastic plasmacytoid dendritic cell neoplasm (BPDCN); Hodgkin's lymphoma; non-Hodgkin's lymphoma (NHL) (including follicular lymphoma (FL), CNS lymphoma, and diffuse large B-cell lymphoma (DLBCL)); light chain amyloidosis; plasma cell leukemia; and multiple myeloma (MM).

[0127] The autoimmune disease in the present invention is preferably an autoimmune disease related to the antigen to which the product used in the present invention binds. Autoimmune disease refers to a condition resulting from an abnormal immune response against normal body cells and body tissues. There are at least 80 types of wide-ranging autoimmune diseases. Some diseases are organ-specific and limited to affecting certain tissues, while others resemble systemic inflammatory diseases that affect many tissues throughout the body. The appearance and severity of these signs and symptoms depend on the location and type of inflammatory response that occurs and may vary over time. Examples of autoimmune diseases that can be treated with the products for use in the present invention described above include rheumatoid arthritis; juvenile dermatomyositis; psoriasis; psoriatic arthritis; lupus; sarcoidosis; Crohn's disease; eczema; nephritis; uveitis; polymyositis; neuritis, e.g., Guillain-Barre syndrome; encephalitis; arachnoiditis; systemic sclerosis; autoimmune-mediated musculoskeletal and connective tissue diseases; neuromuscular degenerative diseases, e.g., Alzheimer's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), neuromyelitis optica, and major, medium and minor glaucoma. These include, but are not limited to, vascular Kawasaki and Henoch-Schönlein vasculitis; cold and warm agglutinin disease; autoimmune hemolytic anemia (AIHA); immune thrombocytopenic purpura (ITP), type 1 diabetes; Hashimoto's thyroiditis; Graves' disease; Graves' ophthalmopathy; adrenalitis; hypophysitis; pemphigus vulgaris; Addison's disease; ankylosing spondylitis; Behcet's syndrome; celiac disease; Goodpasture's syndrome; myasthenia gravis; sarcoidosis; scleroderma; primary sclerosing cholangitis, epidermolysis bullosa acquisita, and pemphigoid.

[0128] The infectious disease in the present invention is preferably an infectious disease associated with the antigen to which the product for use in the present invention binds. Such infectious disease may be bacterial, viral, fungal, parasitic or other infectious disease. Examples of infectious diseases treatable with the product for use in the present invention described above include, but are not limited to, malaria; toxoplasmosis; pneumocystis jirovecii; melioidosis; shigellosis; listeria; diseases caused by Cyclospora or mycobacterium leprae; tuberculosis; and infections in immunocompromised individuals such as HIV positive individuals, individuals on immunosuppressive treatment, or individuals with congenital anomalies such as cystic fibrosis or benign proliferative diseases (e.g. hydatidiform mole or endometriosis).

[0129] The use of the product described herein may be for the manufacture of a medicament described herein. The product described herein for use in the present invention is preferably for a method of treatment, and the product for use is administered in a therapeutically effective amount to a subject, preferably a subject in need thereof. Thus, or in combination with other aspects, in one aspect, the present invention relates to the use of the product of the present invention for the manufacture of a medicament for treating cancer, an autoimmune disease or an infectious disease, in particular cancer. For non-limiting examples of cancer or other diseases that can be treated by the present invention, see the above description.

[0130] Alternatively, in one aspect, in combination with other aspects, the present invention relates to a method for treating cancer, an autoimmune disease or an infectious disease, in particular cancer, comprising administering to a subject in need of said treatment a therapeutically effective amount of a product for use in the present invention. For non-limiting examples of cancer or other diseases that can be treated with the present invention, see above.

[0131] The product used in the present invention is for administration to a subject. The product used in the present invention can be used in the treatment methods described above by administering an effective amount of the composition to a subject in need. In this specification, the term "subject" refers to all animals classified as mammals, including but not limited to primates and humans. The subject is preferably a human. "Therapeutically effective amount" means an amount sufficient to produce a desired response or ameliorate symptoms or signs. The therapeutically effective amount for a particular subject may vary depending on factors such as the condition being treated, the subject's health, the method, route and dose of administration, and the severity of side effects.

[0132] Combination Use In another aspect, the invention provides a product for use in the invention, for use in combination with one or more other therapeutic agents, which may be used simultaneously or sequentially with the one or more other therapeutic agents.

[0133] Suitable chemotherapeutic agents include alkylating agents such as nitrogen mustards, hydroxyurea, nitrosoureas, tetrazines (e.g. temozolomide) and aziridines (e.g. mitomycin); drugs that interfere with the DNA damage response such as PARP inhibitors, ATR and ATM inhibitors, CHK1 and CHK2 inhibitors, DNA-PK inhibitors, and WEE1 inhibitors; antimetabolites such as antifolates (e.g. pemetrexed), fluoropyrimidines (e.g. gemcitabine), deoxynucleoside analogues and thiopurines; microtubule inhibitors such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; cytotoxic antibiotics such as anthracyclines and bleomycin; hypomethylating agents such as decitabine and azacytidine; histone deacetylase inhibitors; all-trans retinoic acid; and arsenic trioxide. Suitable radiotherapeutic agents include radioisotopes such as 131 I-Metaiodobenzylguanidine (MIBG), as sodium phosphate 32 P, chloride 223 Ra, chloride 89 Sr and 153and Sm diamine tetramethylene phosphonate (EDTMP).Suitable substances for use as hormone therapeutic agents include inhibitors of hormone synthesis, such as aromatase inhibitors and GnRH analogues; hormone receptor antagonists, such as selective estrogen receptor modulators (e.g., tamoxifen and fulvestrant) and antiandrogens, such as bicalutamide, enzalutamide and flutamide; CYP17A1 inhibitors, such as abiraterone; and somatostatin analogues.

[0134] Targeted therapeutic agents are therapeutic agents that interfere with specific proteins involved in tumor formation and growth, and may be small molecule drugs; proteins, such as therapeutic antibodies; peptides and peptide derivatives; or protein-small molecule hybrids, such as ADCs. Examples of targeted small molecule drugs include TLR ligands, mTor inhibitors, such as everolimus, temsirolimus, and rapamycin; kinase inhibitors, such as imatinib, dasatinib, and nilotinib; VEGF inhibitors, such as sorafenib and regorafenib; EGFR / HER2 inhibitors, such as gefitinib, lapatinib, and erlotinib; and CDK4 / 6 inhibitors, such as palbociclib, ribociclib, and abemaciclib. Examples of peptide or peptide derivative targeted therapeutic agents include proteasome inhibitors, such as bortezomib and carfilzomib.

[0135] Suitable anti-inflammatory drugs include D-penicillamine, azathioprine and 6-mercaptopurine, cyclosporine, anti-TNF biologics (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab, or certolizumab pegol), lenflunomide, abatacept, tocilizumab, anakinra, ustekinumab, rituximab, daratumumab, ofatumumab, obinutuzumab, secukinumab, apremilast, acetretin, and JAK inhibitors (e.g., tofacitinib, baricitinib, or upadacitinib).

[0136] Immunotherapeutics include substances that induce, enhance or suppress immune responses, such as cytokines (IL-2 and IFN-α); immunomodulatory imid drugs, such as thalidomide, lenalidomide, pomalidomide or imiquimod; therapeutic cancer vaccines, such as talimogene laherparepvec; cell-based immunotherapeutics, such as dendritic cell vaccines, adoptive T cells or chimeric antigen receptor modified T cells; and therapeutic (bispecific) antibodies or other ADCs that can trigger antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) via the Fc region when bound to a membrane-bound ligand on a cell.

[0137] In the present invention, treatment is preferably the prevention, reversal, cure, remission and / or delay of cancer, an autoimmune disease or an infectious disease, which may mean that the severity of at least one symptom of the cancer, an autoimmune disease or an infectious disease is reduced and / or at least one parameter associated with the cancer, an autoimmune disease or an infectious disease is improved.

[0138] In the present invention, the subject may be a survivor and / or considered disease-free. Alternatively, the disease or condition may be stopped or delayed. In the present invention, improved quality of life and observed pain relief may mean that the subject may require less analgesic medication than at the start of treatment. In this context, "less" may mean 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less. The subject may no longer require analgesic medication. These improved quality of life and observed pain relief may be seen, detected, or assessed in a subject after at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more of treatment, and may be compared to the quality of life and observed pain relief at the start of treatment of said subject.

[0139] General definition The conjugates and linker-drugs of the present invention may have one or more chiral centers and / or double bonds and therefore may exist as stereoisomers, e.g., double bond isomers (i.e., geometric isomers), positional isomers, enantiomers or diastereomers. Thus, the chemical structures shown in the specification encompass all possible enantiomers and stereoisomers of the exemplified and specified compounds, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) as well as enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. Compounds may exist in several tautomeric forms, including enol forms, keto forms and mixtures thereof. Thus, the chemical structures shown in the specification encompass all possible tautomeric forms of the exemplified and specified compounds. It is also understood that some isomeric forms, such as diastereomers, enantiomers and geometric isomers, can be separated by physical and / or chemical methods by those skilled in the art. Those skilled in the art will understand that there are chiral centers from the structural formula or substance name, but when chirality is not indicated, all three are individually referred to for each chiral center: racemic mixture, pure R enantiomer and pure S enantiomer. When the structure of a compound is shown as a specific enantiomer, it should be understood that this invention is not limited to that specific enantiomer. When two moieties are bonded together, this means that these moieties do not exist as atoms, and valence compliance is met by replacing electronic bonds. All of these are known in the art.

[0140] The compounds disclosed in this specification and claims may exist as exo and endo regioisomers. Unless otherwise stated, the description of a compound in the specification and claims is intended to include both the individual exo and individual endo regioisomers of the compound, as well as mixtures thereof. Furthermore, the compounds disclosed in this specification and claims may exist as cis and trans isomers. Unless otherwise stated, the description of a compound in the specification and claims is intended to include both the individual cis and individual trans isomers of the compound, as well as mixtures thereof. For example, if the structure of a compound is shown as a cis isomer, it should be understood that the corresponding trans isomer, or mixtures of cis and trans isomers, are not excluded from this invention.

[0141] In this document and the claims thereto, the verb "comprise" and its conjugations are used in its open-ended sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that there is more than one of that element, unless the context clearly requires that there is one and only one of that element. Thus, the indefinite article "a" or "an" usually means "at least one."

[0142] "About" or "approximately" (eg, about 10), when used in conjunction with a numerical value, preferably means that the value may be 1% more or less than the given value.

[0143] When parameters of a substance are discussed in this invention, it is assumed that the parameters are determined, measured, or determined at physiological conditions, unless otherwise specified. Physiological conditions are known to those of skill in the art and include aqueous solvent systems, atmospheric pressure, a pH of 6-8, room temperature (RT) to about 37° C. (about 20° C. to about 40° C.), and appropriate concentrations of buffer salts or other components. It is understood that charge often involves balance. A moiety that is said to carry or possess a charge is one that is found in a state in which it possesses or possesses such charge more frequently than in a state in which it does not possess or possess such charge. As such, as will be understood by those of skill in the art, atoms shown in this specification to be charged may be uncharged under certain conditions, and neutral moieties may be charged under certain conditions.

[0144] All patents and articles cited in this specification are hereby incorporated by reference in their entirety.

[0145] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. EXAMPLES

[0146] General Procedure solvent : Reagent grade or HPLC grade solvents from a variety of vendors were used. NMR spectra : NMR spectra were obtained using a Bruker AVANCE400 ( 1 400MHz for H; 13 C, 101 MHz). Chemical shift : Chemical shifts are given in ppm relative to the internal standard tetramethylsilane or residual non-deuterated solvent. UPLC measurement of products : The products were measured on a Waters UPLC-MS (with SQD 2 detector) equipped with a Waters ACQUITY UPLC BEH C18 column (1.7 μm particle size, 2.1 × 50 mm) at a flow rate of 0.4 mL / min (MeCN / water × 0.1% formic acid). HPLC purification : Preparative HPLC purification was performed on a Shimadzu Prominence 20AP system equipped with a Waters SunFire Prep C18 OBD 5 μm column (19×150 mm) at a flow rate of 17 ml / min.

[0147] General Procedure XXA: Alkylation of Alcohols with Chloromethyl Carbamates Paraformaldehyde (3 equiv.) and trimethylsilyl chloride (TMSCl) (2.75 equiv.) were added sequentially to a suspension (room temperature) of carbamate (2.5 equiv.) in dichloromethane (DCM) (0.45 M carbamate) under nitrogen. The mixture was stirred for 1 h, then concentrated, coevaporated with DCM, and dried under high vacuum for 2 min. The pale yellow oil was dissolved in DCM (0.6 M carbamate).

[0148] An aliquot of this solution (usually 1.5 equiv. of chloromethyl carbamate) was then added to a cooled (0 °C) mixture of alcohol (1 equiv.) in DCM (0.24 M). N,N-Diisopropylethylamine (DIPEA, 3 equiv.) was added and after stirring for 5 min, the reaction was allowed to warm to room temperature. After 30-60 min, conversion was assessed using UPLC-MS and, if incomplete, further stock solution was added. With each addition of chloromethyl carbamate, a stoichiometric amount of DIPEA was added to maintain a basic pH. When complete conversion was observed, the reaction was quenched with MeOH, concentrated, and purified.

[0149] General Procedure XXB: Preparation of Phosphonic Acid Dichlorides from Phosphonate Diesters Trimethylsilyl bromide (TMSBr, 10 equiv.) was added to a cooled (0° C.) solution of the phosphonate diester (1 equiv.) in DCM (0.2 M) under nitrogen atmosphere over 5 min. The ice bath was removed after 30 min and the reaction was stirred at room temperature for 3.5 h. The solution was concentrated using a rotary evaporator purged with nitrogen gas and the crude product was dissolved in DCM (0.2 M) under nitrogen atmosphere and cooled to 0° C. Dimethylformamide (DMF, 2 drops) was added followed by the dropwise addition of oxalyl chloride (3 equiv.). The cooling bath was allowed to warm to room temperature over 1 h and stirred for 16 h. The reaction was concentrated under nitrogen atmosphere and co-evaporated with DCM (3× 10 mL) to give the crude phosphonic acid dichloride which could be used without further purification.

[0150] General Procedure XXC: Allylic Oxidation with SeO2 Step 1 SeO2 (0.7 equiv.) and salicylic acid (0.1 equiv.) were dissolved in DCM (0.9 M SeO2) and t-BuOOH (4.5 equiv.) was added at room temperature. After vigorous stirring for 15 min, alkene (1 equiv.) in DCM (0.82 M) was added. The resulting reaction mixture was vigorously stirred until UPLC-MS analysis indicated complete consumption of the alkene (usually 16-48 h). The reaction mixture was cooled to 0 °C and then carefully quenched with saturated aqueous NaHCO3 (10 mL per mL of tBuOOH used). The mixture was diluted with water to help dissolve precipitated salts and the product was extracted 3-6 times with DCM (or EtOAc for more polar compounds) until no product was present in the aqueous phase by UPLC-MS analysis. The combined organic layers were dried over Na2SO4, filtered and concentrated to give a mixture of allylic alcohols and the corresponding aldehyde products.

[0151] Step 2 The crude product was dissolved in EtOAc (0.2 M) and AcOH (5 equiv.) was added followed by NaBH(OAc)3 (5 equiv.). The reaction mixture was stirred at 50° C. until UPLC-MS analysis showed complete consumption of the aldehyde (usually 1-3 h). The reaction mixture was then cooled to room temperature and water (1-5 vol.) was added. The aqueous layer was extracted with EtOAc (2-6×) until no product was present in the aqueous phase by UPLC-MS analysis. The combined organic layers were washed with a small amount of saturated aqueous NaHCO3 and brine, dried over Na2SO4, filtered, concentrated and purified.

[0152] Example 1 Synthesis of carbamate (XD5) [ka]

[0153] (S)-2-Azido-N-(4-(hydroxymethyl)phenyl)propanamide (XD7) (S)-2-Azidopropionic acid (6.73 g, 58.5 mmol) and (4-aminophenyl)methanol (10.0 g, 82 mmol) were dissolved in DCM (228 mL) and MeOH (75 mL). After cooling to 0 °C, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 28.9 g, 117 mmol) was added and the mixture was stirred at room temperature overnight and concentrated. Flash chromatography (silica gel, 0-40% EtOAc in DCM) afforded the azide XD7 (9.3 g, 72%) as a yellow liquid. MS (ESI + ) C 10 H 13 N4O2 + [M+H] + Calculated value: 221.10, actual value: 221.11.

[0154] Ethyl carbamic acid (S)-4-(2-azidopropanamide) benzyl ester (XD5) To a solution of XD7 (3.8 g, 17.2 mmol) in tetrahydrofuran (THF, 100 mL) at 0° C., dibutyltin dilaurate (2.57 mL, 4.31 mmol) and ethyl isocyanate (2.05 mL, 25.9 mmol) were added and the mixture was stirred at room temperature for 5 h. The reaction mixture was concentrated onto silica gel and purified by flash chromatography (silica gel, 0-50% diethyl ether in heptane, followed by 0-100% EtOAc in heptane) to give the azide XD5 (4.25 g, 85%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 8.29 (br s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 5.04 (s, 2H), 4.92 (br s, 1H), 4.18 (q, J = 7.0 Hz, 1H), 3.22 (quint, J = 6.7 Hz, 2H), 1.61 (d, J = 7.0 Hz, 3H), 1.12 (t, J = 7.3 Hz, 3H). MS (ESI + ) C 13 H 17 N5NaO3 + [M+Na] + Calculated value: 314.1, actual value: 314.1.

[0155] Example 2 Synthesis of Linker-Drug Compound XD4 from pAg (Prodrug) Moiety XD1 [ka]

[0156] (((E)-5-(((((4-((S)-2-azidopropanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XD2) XD1 (100 mg, 0.240 mmol, prepared as described in Kadri et al., J. Med. Chem. 2020, 63, 11258-11270) was reacted with the carbamate XD5 according to general procedure XXA. Flash chromatography (silica gel, 0-5% MeOH in DCM) afforded XD2 (134 mg, 78%) as a colorless oil. MS (ESI + ) C 36 H 46 N6O8P + [M+H] + Calculated value: 721.3, actual value: 721.6.

[0157] (((E)-5-(((((4-((S)-2-aminopropanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XD3) A solution of azide XD2 (134 mg, 0.186 mmol) in THF / water (2 mL, 9:1) was purged with nitrogen gas for 15 min. Tributylphosphine (0.116 ml, 0.465 mmol) was added at room temperature and the mixture was stirred at room temperature for 4 h. The reaction was concentrated and the water content was removed by coevaporation with MeCN (2× 7 mL) and toluene (1× 7 mL). The crude product was purified by flash chromatography (silica gel, 0–20% MeOH in DCM) to give the amine XD3 (97 mg, 75%). MS (ESI + ) C 36 H 48 N4O8P + [M+H] + Calculated value: 695.3, actual value: 695.5.

[0158] (((E)-5-(((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XD4) To a suspension of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (26.5 mg, 0.085 mmol, prepared as described in WO 2013 / 122823), DMAP (1.0 mg, 8.5 μmol) and N-hydroxyphthalimide (13.9 mg, 0.085 mmol) in THF (0.8 mL) at room temperature was added N,N'-diisopropylcarbodiimide (DIC; 0.013 mL, 0.085 mmol). After 3 h at room temperature, the reaction mixture was concentrated and suspended in DCM (~1 mL). The orange / red supernatant was then added to a solution of XD3 (41 mg, 0.059 mmol) in DMF (0.5 mL) and stirred at room temperature for 75 min. After concentration, the crude product was purified by RP-HPLC (water / MeCN, 40% to 90% gradient, no modifiers were used). Lyophilization of the product fractions afforded XD4 (10.9 mg, 13%). MS (ESI + ) C 51 H 68 NO 12 P + [M+H] + Calculated value: 987.5, actual value: 987.7.

[0159] Linker-drug compound XD4 was conjugated to the antibody to prepare conjugates ADC-XD4-r and ADC-XD4-i as described in Example 22. Their effects on γδ T cells were tested as described in Example 23.

[0160] Example 3 Synthesis of Linker-Drug Compound XD13 [ka]

[0161] P-(But-3-en-1-yl)-N-(cyclobutylmethyl)phosphonamidate 4-((S)-2-azidopropanamide) benzyl ester (XD9) First, but-3-en-1-ylphosphonic acid dichloride (XD8, 338 mg, 1.95 mmol, prepared as described in Kadri et al. J. Med. Chem. 2020, 63, 11258-11270) was added dropwise to a solution of cyclobutanemethanamine (166 mg, 1.95 mmol) and Et3N (0.544 ml, 3.90 mmol) in DCM (3.8 mL) at -78 ° C. The cooling bath was removed after 5 min and stirring was continued for 45 min.

[0162] In a separate flask, alcohol XD7 (429 mg, 1.95 mmol) and Et3N (0.544 ml, 3.90 mmol) were dissolved in DCM (3.8 mL) under nitrogen atmosphere and the mixture was cooled to -78 °C. The solution prepared in the first step was then filtered and added directly to the solution containing alcohol XD7. The transfer was completed using DCM (2 mL). After 5 min, the reaction was warmed to room temperature and stirred for 5 h. The reaction was quenched with 1-methylpiperazine (0.1 mL). After concentration, the crude product was dissolved in EtOAc (50 mL) and washed with hydrochloric acid (0.1 M, 30 mL). The aqueous phase was extracted with EtOAc (15 mL) and the combined organic phase was washed with saturated aqueous NaHCO3, water and brine, dried over MgSO4, filtered and concentrated. Flash chromatography (silica gel, 0-80% EtOAc in DCM) afforded the azide XD9 (363 mg, 46%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 8.21 (br s, 1H), 7.56 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 5.85 (ddt, J = 16.9, 10.3, 6.4 Hz, 1H), 5.09-4.96 (m, 3H), 4.89 (dd, J = 11.9, 7.6 Hz, 1H), 4.24 (q, J = 7.0 Hz, 1H), 2.88 (br s, 2H), 2.43-2.27 (m, 4H), 2.13-1.97 (m, 2H), 1.97-1.75 (m, 4H), 1.70-1.55 (m, 5H). MS (ESI + ) C 19 H29 N5O3P + [M+H] + Calculated value: 406.2, actual value: 406.4.

[0163] P-(But-3-en-1-yl)-N-(cyclobutylmethyl)phosphonamidate 4-((S)-2-aminopropanamide) benzyl ester (XD10) A solution of azide XD9 (244 mg, 0.602 mmol) in THF (1.8 mL) / water (0.2 mL) was purged with nitrogen gas for 15 min. Tributylphosphine (0.376 mL, 1.51 mmol) was added at room temperature and the mixture was stirred for 22 h. The reaction was concentrated and co-evaporated with MeCN (2× 7 mL) and toluene (1× 7 mL) and the crude product was purified by flash chromatography (silica gel, 0-20% MeOH in DCM) to give the amine XD10 (182 mg, 80%). 1 H NMR (400 MHz, CD3OD) ppm = 7.63 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 5.89 (ddt, J = 16.9, 10.3, 6.4 Hz, 1H), 5.07 (dq, J = 17.0, 1.6 Hz, 1H), 5.02-4.89 (m, 3H), 3.58 (q, J = 6.9 Hz, 1H), 2.99-2.85 (m, 2H), 2.50-2.39 (m, 1H), 2.38-2.27 (m, 2H), 2.12-2.01 (m, 2H), 1.98-1.77 (m, 4H), 1.77-1.66 (m, 2H), 1.37 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 19 H 31 N3O3P + [M+H] + Calculated value: 380.2, actual value: 380.3.

[0164] ((2S)-1-(((2S)-1-((4-(((but-3-en-1-yl((cyclobutylmethyl)amino)phosphoryl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (XD11) To a solution of amine XD10 (182 mg, 0.48 mmol) and DIPEA (0.079 ml, 0.46 mmol) in THF (4.8 mL) was added Fmoc-Val-OSu (220 mg, 0.50 mmol) at room temperature. After 70 min a gel-like mixture was formed. Ethyl acetate (4.0 mL) was added to break up the gel and stirring was continued for 4 h. The reaction mixture was diluted with EtOAc / isopropyl alcohol (9:1) and washed with saturated aqueous NaHCO3 and brine. The organic phase was dried over Na2SO4, filtered and concentrated. Flash chromatography (silica gel, 0-10% MeOH in DCM) afforded amide XD11 (279 mg, 83%). 1H NMR (400 MHz, DMSO-d6) ppm = 10.00 (s, 1H), 8.17 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (t, J = 7.3 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.46-7.38 (m, 3H), 7.37-7.27 (m, 4H), 5.87 (ddt, J = 16.9, 10.4, 6.3 Hz, 1H), 5.08-4.91 (m, 2H), 4.84 (dd, J = 12.1, 7.6 Hz, 1H), 4.77 (dd, J = 12.1, 7.6 Hz, 1H), 4.57 (dt, J = 11.1, 6.8 Hz, 1H), 4.43 (quint, J = 7.0 Hz, 1H), 4.34-4.18 (m, 3H), 3.92 (dd, J = 8.9, 7.1 Hz, 1H), 2.87-2.73 (m, 2H), 2.39-2.26 (m, 1H), 2.26-2.15 (m, 2H), 2.05-1.90 (m, 3H), 1.85-1.59 (m, 6H), 1.31 (d, J = 7.1 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 39 H 50 N4O6P + [M+H] + Calculated value: 701.4, actual value: 701.5.

[0165] ((2S)-1-(((2S)-1-((4-(((((cyclobutylmethyl)amino)((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (XD12) Amide XD11 (100 mg, 0.143 mmol), 2-methylprop-2-en-1-ol (0.126 ml, 1.50 mmol) and 1,4-benzoquinone (1.5 mg, 0.014 mmol) were suspended in 1,2-dichloroethane (1.2 mL) at room temperature under nitrogen atmosphere. Hoveyda-Grubbs 2nd Generation Catalyst (4.5 mg, 7.1 μmol, CAS: 301224-40-8) was added at room temperature and the suspension was heated to 45°C. After 4 hours, an additional 4.5 mg, 7.1 μmol of Hoveyda-Grubbs 2nd Generation Catalyst was added and stirring was continued at 45°C overnight. An additional 2.3 mg, 0.021 mmol) of 1,4-benzoquinone and Hoveyda-Grubbs 2nd Generation Catalyst (8.9 mg, 0.014 mmol) were added and the reaction was continued for 5 hours. The reaction was cooled to room temperature, 1,4-bis(3-isocyanopropyl)piperazine (SnatchCat, 12.6 mg, 0.057 mmol) was added, and the mixture was stirred for 30 min. Flash chromatography (silica gel, 0-10% MeOH in DCM) afforded XD12 (39 mg, contaminated with an impurity at the internal position (m / z 731.6) from the undesired Z isomer and double bond isomerization in the sm prior to cross-metathesis). This material was used in the next step without further purification. MS (ESI + ) C 41 H 54 N4O7P + [M+H] + Calculated value: 745.4, actual value: 745.6.

[0166] N-(cyclobutylmethyl)-P-((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl ester (XD13) Dipeptide XD12 (39 mg, 0.052 mmol) was dissolved in DMF (1 mL) at room temperature. Piperidine (0.39 ml, 3.9 mmol) was added and the mixture was stirred for 30 min. After concentration, ether (8 mL) was added and the mixture was stirred for 15 min at room temperature. The product did not fully dissolve and stuck to the flask. The ether was removed by pipetting and the flask was rinsed with ether (1×). The remaining oil was dried in vacuum to give a colorless oil (24.5 mg).

[0167] This material was dissolved in DMF (0.5 mL) at room temperature, and 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (14.5 mg, 0.047 mmol) and DIPEA (0.025 mL, 0.141 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 3 h. After concentration, the crude product was purified by RP-HPLC (water / MeCN, 70:30 to 50:50 gradient, no modifiers used) to give pure XD13 (16.2 mg, 43%, 2 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 9.91 (s, 1H), 8.13 (d, J = 7.0 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.62-7.56 (m, 2H), 7.31 (d, J = 8.5 Hz, 2H), 7.00 (s, 2H), 5.35 (td, J = 7.2, 1.3 Hz, 1H), 4.83 (dd, J = 12.3, 7.6 Hz, 1H), 4.76 (dd, J = 12.1, 7.8 Hz, 1H), 4.63 (t, J = 5.6 Hz, 1H), 4.54 (dt, J = 11.0, 6.8 Hz, 1H), 4.39 (quint, J = 7.0 Hz, 1H), 4.17 (dd, J = 8.6, 6.9 Hz, 1H), 3.76 (d, J = 5.8 Hz, 2H), 3.36 (t, J = 7.1 Hz, 2H), 2.88-2.72 (m, 2H), 2.39-2.26 (m, 1H), 2.23-2.10 (m, 4H), 2.02-1.88 (m, 3H), 1.87-1.70 (m, 2H), 1.69-1.57 (m, 4H), 1.56-1.52 (m, 3H), 1.52-1.40 (m, 4H), 1.30 (d, J = 7.1 Hz, 3H), 1.18 (quint, J = 7.5 Hz, 2H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H). MS (ESI + ) C 36 H 55 N5O8P + [M+H] + Calculated value: 716.4, actual value: 716.6.

[0168] Linker-drug compound XD13 was conjugated to the antibody to prepare conjugates ADC-XD13-r and ADC-XD13-i as described in Example 22. Their effects on γδ T cells were tested as described in Example 23.

[0169] Example 4 Synthesis of Carboxylic Acid XT2 [ka]

[0170] ((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)carbonyl)-L-valine (XT2) To a solution of L-valine (167 mg, 1.4 mmol) and carbonate ester XT1 (500 mg, 1.4 mmol, prepared as described in Elgersma et al., Mol. Pharm., 2015, 12, 1813-1835) in DMF (5 mL) at 0 °C, DIPEA (0.249 ml, 1.40 mmol) was added and the resulting mixture was stirred at room temperature for 10 days. The mixture was concentrated, dissolved in EtOAc (25 mL) and washed with hydrochloric acid (1 M, 50 mL). The aqueous phase was extracted with EtOAc (25 mL) and the combined organic phase was dried (MgSO4), filtered and concentrated. Flash chromatography (silica gel, 0-40% MeOH in DCM) afforded the acid XT2 (250 mg, 53%) as a clear oil. MS (ESI + ) C 14 H 21 N2O7 + [M+H] + Calculated value: 329.1, actual value: 329.2.

[0171] Example 5 Synthesis of Linker-(Pro)drug Compound XC4 [ka]

[0172] (S,E)-(((5-(((((4-(2-azidopropanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoic acid) ester (XC2) Alcohol XC1 (70 mg, 0.171 mmol, prepared as described in Wiemer, Chem. Biol. 2014, 21, 945-954) was reacted with carbamate XD5 according to general procedure XXA described in Example 1. After completion of the reaction, half of the solvent was removed by rotary evaporation. The crude mixture was then loaded directly onto a silica gel column and purified by flash chromatography (silica gel, 0-100% EtOAc in heptane). Azide XC2 (140 mg, quantitative) was obtained as an impure colorless oil and was used in the next step without further purification. MS (ESI + ) C 32 H 51 N5O 11 P + [M+H] + Calculated value: 712.3, actual value: 712.5.

[0173] (S,E)-(((5-(((((4-(2-aminopropanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoic acid) ester (XC3) To a solution of azide XC2 (70 mg, 0.098 mmol) in THF (1.85 mL) / water (0.093 mL) at room temperature was added tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl, 85 mg, 0.295 mmol), and the resulting mixture was stirred for 18 h. The suspension was filtered through cotton wool, rinsed with THF, and the filtrate was concentrated onto silica gel. Flash chromatography (silica gel, 0–10% MeOH in DCM) afforded the amine XC3 (15 mg, 22%) as a colorless oil. 1H NMR (400 MHz, CD3OD) ppm = 7.51 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 5.61-5.52 (m, 4H), 5.35-5.13 (m, 1H), 5.02 (s, 2H), 4.65 (br s, 2H), 3.80 (q, J = 7.0 Hz, 1H), 3.70 (br d, J = 1.0 Hz, 2H), 3.27 (q, J = 7.0 Hz, 2H), 2.27-2.09 (m, 2H), 1.91-1.71 (m, 2H), 1.58-1.45 (m, 3H), 1.42 (d, J = 7.0 Hz, 3H), 1.13 (s, 18H), 1.09-1.00 (m, 3H). MS (ESI + ) C 32 H 53 N3O 11 P + [M+H] + Calculated value: 686.3, actual value: 686.7.

[0174] ((((E)-5-(((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7-dioxo-8,11-dioxa-3,6-diazatridecanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoic acid) ester (XC4) To the amine XC3 (15 mg, 0.022 mmol) was added a solution of the acid XT2 (7.2 mg, 0.022 mmol) in DMF (0.500 mL). The mixture was cooled in an ice bath and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU, 10.0 mg, 0.026 mmol) and DIPEA (7.6 μl, 0.044 mmol) were added sequentially and the resulting mixture was stirred while gradually warming to room temperature. After stirring at room temperature for 1.5 h, the reaction was concentrated in vacuo and the crude product was purified by flash chromatography (silica gel, 0-8% MeOH in DCM) to give XC4 (18 mg) contaminated with XT2. The product was dissolved in EtOAc and then washed with saturated aqueous NaHCO3 / water (1:1), water and brine. The organic phase was dried over Na2SO4, filtered, concentrated and further purified by flash chromatography (silica gel, 0-8% MeOH in DCM) to give XC4 (10 mg, 45%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 8.80-8.51 (m, 1H), 7.65-7.52 (m, 2H), 7.33-7.28 (m, 2H), 7.14-6.94 (m, 1H), 6.70 (s, 2H), 5.67 (d, J = 12.9 Hz, 4H), 5.39-5.27 (m, 1H), 5.16 (br s, 1H), 5.09 (s, 2H), 4.77 (br s, 1H), 4.74-4.63 (m, 2H), 4.30 (br s, 1H), 4.09 (br s, 1H), 4.03 (br t, J = 5.5 Hz, 1H), 3.84 (br s, 1H), 3.76-3.60 (m, 5H), 3.55 (br s, 2H), 3.44-3.29 (m, 2H), 2.38-2.18 (m, 3H), 1.98-1.70 (m, 2H), 1.65-1.53 ​​(m, 3H), 1.46 (br d, J = 7.0 Hz, 3H), 1.23 (s, 18H), 1.18-1.10 (m, 3H), 1.01 (br d, J = 6.6 Hz, 3H), 0.96 (br d, J = 6.9 Hz, 3H). MS (ESI + ) C 46 H 71 N5O 17 P + [M+H] + Calculated value: 996.5, actual value: 996.7.

[0175] Linker-drug compound XC4 was conjugated to the antibody to prepare conjugates ADC-XC4-r and ADC-XC4-i as described in Example 22. Their effects on γδ T cells were tested as described in Example 23.

[0176] Example 6 Preparation of Linker-Drug Compound XD18

[0177] [ka]

[0178] (4-Methylpent-3-en-1-yl)phosphonic acid dimethyl ester (XD15) To a stirred solution of diisopropylamine (13.0 ml, 92.7 mmol) in THF (280 mL) at -78 °C was added n-butyllithium (1.6 M in hexanes, 55.4 ml, 88.6 mmol). The resulting solution was stirred at -78 °C for 20 min. Methylphosphonic acid dimethyl ester (8.73 ml, 80.6 mmol) was then added slowly using a syringe and the mixture was stirred for 1 h. Prenyl bromide (11.6 ml, 101 mmol) was added slowly using a syringe and the solution was then allowed to warm to room temperature overnight. The reaction was cooled on an ice bath, quenched with saturated aqueous NH4Cl, and extracted with Et2O (3x). The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated. Purification was performed by flash chromatography (silica gel, 0-3% MeOH in ether). The product fractions were concentrated and co-evaporated with DCM (3x) to remove traces of methanol to give the phosphonate diester XD15 (12.88 g, 83%) as a yellow liquid. 1 H NMR (400 MHz, CDCl3) ppm = 5.14-5.07 (m, 1H), 3.74 (d, J = 10.8 Hz, 6H), 2.33-2.22 (m, 2H), 1.83-1.71 (m, 2H), 1.69 (s, 3H), 1.62 (s, 3H). MS (ESI + ) C8H 18 O3P + [M+H] + Calculated value: 193.1, actual value: 193.1.

[0179] ((2S)-1-(((2S)-1-((4-((((2-cyanoethoxy)(4-methylpent-3-en-1-yl)phosphoryl)-oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (XD16) Step 1 To (4-methylpent-3-en-1-yl)phosphonic acid dichloride (97.0 mg, 0.485 mmol, prepared from phosphonic acid diester XD15 according to general procedure XXB) in DCM (1.8 mL) was added 5-(ethylthio)-1H-tetrazole (6.31 mg, 0.048 mmol). The solution was cooled to -78°C and 3-hydroxypropanenitrile (0.033 ml, 0.485 mmol) and pyridine (0.047 ml, 0.582 mmol) were added. After stirring at -78°C for 30 min, the reaction was allowed to warm to room temperature and stirred for 2.5 h.

[0180] Step 2 In a separate flask, Fmoc-Val-Ala-PAB (250 mg, 0.485 mmol) was dissolved in pyridine (1.0 mL). After cooling to 0° C., the DCM solution of phosphonic chloride prepared in step 1 was added dropwise to the pyridine solution using a cannula. The reaction was stirred at 0° C. for 30 min and then at room temperature for 1 h. UPLC-MS analysis showed that the reaction had stopped at 50% conversion. The reaction was stored at −30° C. overnight and then repeated with the same volume, changing the 2.5 h of stirring in step 1 to overnight stirring at room temperature. The next day, the resulting solution was added to the reaction mixture stored overnight at 0° C. After stirring at 0° C. for 30 min and at room temperature for 1 h, the reaction was observed to be completely complete. The solution was concentrated and the crude product was dry loaded onto silica gel and purified by flash chromatography (silica gel, 0-25% acetone in DCM) to give the phosphonate diester mixture XD16 (127 mg, 37%) as an impure white solid. MS (ESI+) C 39 H 48 N4O7P + [M + H] + Calculated value: 715.3, actual value: 715.5.

[0181] ((2S)-1-(((2S)-1-((4-((((2-cyanoethoxy)((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (XD17) SeO2 (71 mg, 0.64 mmol) and 2-hydroxybenzoic acid (17 mg, 0.12 mmol) were dissolved in DCM (0.7 mL) and t-BuOOH (70% in water, 0.66 ml, 4.81 mmol) was added at room temperature. After vigorous stirring for 15 min, the solution was pipetted into a suspension of XD16 (127 mg, 0.178 mmol) in DCM (1.1 mL). The resulting mixture was vigorously stirred overnight. The mixture was cooled to 0 °C and slowly quenched with saturated aqueous NaHCO3 until effervescence ceased. Water was added to dissolve the precipitated salts. The product was extracted with DCM (3x) and the combined organic phase was dried over Na2SO4 and concentrated onto silica gel. Flash chromatography (silica gel, 0-5% MeOH in DCM) afforded the alcohol XD17 (45 mg, 35%) as a pure white solid which could be used in the next step. MS (ESI+) C 39 H 48 N4O8P + [M+H] + Calculated value: 731.3, actual value: 731.6.

[0182] ((E)-5-Hydroxy-4-methylpent-3-en-1-yl)phosphonic acid 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido) benzyl ester (XD18) Step 1 Phosphonate ester XD17 (45 mg, 0.062 mmol) in THF (1.0 mL) was diluted with MeOH (9.0 mL). A solution of ammonia in methanol (7 M, 2.35 mL) was then added at room temperature and the mixture was stirred at room temperature for 3 h. Aqueous NaOH (2 M, 1.15 mL) was then added at room temperature and the mixture was stirred for 15 min. The reaction was cooled on an ice bath and aqueous AcOH (1 M, 23.5 mL) was added. A cloudy solution was obtained and filtered through a syringe filter. The filtrate was concentrated in vacuo and dissolved in dioxane / water (1:1, 1 mL). The solution was lyophilized to give 200 mg of a white solid (mixture of product and salt).

[0183] Step 2 The product was dissolved in DMF (1 mL), DIPEA (0.049 mL, 0.281 mmol) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (70.4 mg, 0.228 mmol) were added at room temperature, and the mixture was stirred for 30 min. The excess base was quenched with aqueous AcOH (1 M, 0.52 mL) at 0 °C, and the mixture was concentrated. The crude product was purified by preparative RP-HPLC (water x 0.1% TFA / MeCN x 0.1% 2,2,2-trifluoroacetic acid (TFA) / MeCN, 90:10 to 45:55 gradient). MeCN was removed on a rotary evaporator, and the aqueous mixture was lyophilized to give XD18 (26.5 mg, 89% for two steps). 1H NMR (400 MHz, DMSO-d6) ppm = 9.92 (s, 1H), 8.14 (d, J = 7.0 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 6.99 (s, 2H), 5.33 (td, J = 7.1, 1.0 Hz, 1H), 4.84 (br d, J = 7.6 Hz, 2H), 4.61 (br s, 1H), 4.39 (quint, J = 6.9 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 3.74 (s, 2H), 3.36 (t, J = 7.0 Hz, 2H), 2.26-2.06 (m, 4H), 2.02-1.91 (m, 1H), 1.66-1.54 (m, 2H), 1.50 (s, 3H), 1.51-1.39 (m, 4H), 1.30 (d, J = 7.1 Hz, 3H), 1.26-1.12 (m, 3H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 31 H 46 N4O9P + [M+H] + The calculated value is 649.3 and the measured value is 649.6.

[0184] Example 7 Synthesis of Rincon compound XC9

[0185]

change

[0186] (4-ニトロフェニル) (S)-4-(2-アジドプロパンアミド)ベンジルエステル carbonate (XC5) To a solution of alcohol XD7 (1.60 g, 7.27 mmol) in THF (20 mL) at 0 °C was added bis(4-nitrophenyl)carbonate (4.42 g, 14.5 mmol) and DIPEA (1.90 mL, 10.9 mmol). The resulting mixture was stirred at room temperature for 18 h and then concentrated in vacuo. Flash chromatography (silica gel, 0-5% EtOAc in DCM) afforded carbonate XC5 (1.97 g, 70%) as a pale yellow oil. MS (ESI + ) C 17 H 16 N5O6 + [M+H] + Calculated value: 386.1, actual value: 386.2.

[0187] (2,5,8,11,14,17,20-Heptaoxadocosan-22-yl)carbamic acid (S)-4-(2-azidopropanamide) benzyl ester (XC6) To a solution of carbonate XC5 (624 mg, 1.62 mmol) in THF (10.8 mL) at 0 °C, 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (550 mg, 1.62 mmol) and DIPEA (0.340 mL, 1.94 mmol) were added and the resulting bright yellow solution was stirred for 18 h while gradually warming to room temperature. After concentration, the crude product was purified by flash chromatography (silica gel, 0-6% MeOH in DCM) to give carbamate XC6 (875 mg, 92%) as a pale yellow oil. MS (ESI + ) C 26 H 44 N5O 10 + [M+H] + Calculated value: 586.3, actual value: 586.4.

[0188] (((E)-23-(((4-((S)-2-azidopropanamido)benzyl)oxy)carbonyl)-27-methyl-2,5,8,11,14,17,20,25-octaoxa-23-azatriacont-27-en-30-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XC7) Alcohol XD1 (100 mg, 0.240 mmol, prepared as described in Kadri, H. et al. J. Med. Chem. 2020, 63, 11258-11270) was reacted with carbamate XC6 according to general procedure XXA. Flash chromatography (silica gel, 0-11% MeOH in DCM) afforded impure azide XC7 (313 mg) as a colorless oil, which was used in the next step without further purification. MS (ESI + ) C 49 H 71 N6NaO 15 P + Calculated value: 1037.5, actual value: 1037.7.

[0189] (((E)-23-(((4-((S)-2-aminopropanamido)benzyl)oxy)carbonyl)-27-methyl-2,5,8,11,14,17,20,25-octaoxa-23-azatriacont-27-en-30-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XC8) A solution of azide XC7 (121 mg, 0.119 mmol) in THF (1.14 mL) / water (0.13 mL) was purged with nitrogen gas for 15 min. Tributylphosphine (0.074 ml, 0.298 mmol) was added at 0° C. and the mixture was stirred at room temperature for 5.5 h and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 0-15% MeOH in DCM) to give amine XC8 (62 mg, 52%) as a yellow oil. MS (ESI + ) C 49 H 74 N4O 15 P + [M+H] + Calculated value: 989.5, actual value: 989.8.

[0190] (((E)-23-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-27-methyl-2,5,8,11,14,17,20,25-octaoxa-23-azatriacont-27-en-30-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XC9) To a cooled (0 °C) solution of amine XC8 (62 mg, 0.063 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (19.5 mg, 0.063 mmol, prepared as described in WO 2013 / 122823) in DMF (1 mL) was added HATU (28.6 mg, 0.075 mmol) and DIPEA (0.022 mL, 0.125 mmol). The resulting yellow mixture was stirred at room temperature for 1.5 h and concentrated in vacuo. The residue was dissolved in EtOAc, washed with saturated aqueous NaHCO3 / water (1:1), water and brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0-10% MeOH in DCM) to give a colorless oil. The oil was dissolved in MeCN / MilliQ water (1:1) and purified by preparative RP-HPLC (water / MeCN, 60:40 to 10:90 gradient, no modifiers used). Product fractions were pooled and MeCN was removed by rotary evaporation. The aqueous mixture was then lyophilized to give maleimide XC9 (25 mg, 31%) as a colorless oil. MS (ESI + ) C 64 H 94 NO 19 P + [M+H] + Calculated value: 1281.6, actual value: 1282.0.

[0191] Example 8 Synthesis of Linker-Drug Compound XC13 Preparation of (E)-((5-hydroxy-4-methylpent-3-en-1-yl)(4-iodophenoxy)phosphoryl)alanine benzyl ester (XS4) [ka]

[0192] ((4-iodophenoxy)(4-methylpent-3-en-1-yl)phosphoryl)alanine benzyl ester (XS3) To (4-methylpent-3-en-1-yl)phosphonic acid dichloride (837 mg, 4.16 mmol, prepared from phosphonic acid diester XD15 (1.00 g, 5.20 mmol) according to general procedure XXB) in toluene (27 mL) was added dropwise a solution of 4-iodophenol (1.83 g, 8.33 mmol) and DIPEA (1.45 mL, 8.33 mmol) in toluene (27 mL) at -78 °C. The reaction mixture was stirred for 30 min at -78 °C. L-alanine benzyl ester hydrochloride (1.89 g, 8.74 mmol) and DIPEA (3.05 mL, 17.5 mmol) were added and the reaction mixture was allowed to come to room temperature and stirred for 2 h. The reaction mixture was concentrated and the crude product was purified by flash chromatography (silica gel, 0 to 50% EtOAc in heptane) to give XS3 (0.490 g, 22%, ca. 3:2 diastereomeric mixture) as a yellow solid. 1 H NMR (400 MHz, CDCl3) ppm = 7.60-7.53 (m, 2H), 7.41-7.28 (m, 5H), 7.00-6.93 (m, 2H), 5.14-5.06 (m, 3H), 4.15-4.04 (m, 1H), 3.31 (t, J = 10.4 Hz, 0.6H), 3.22 (t, J = 10.5 Hz, 0.4H), 2.41-2.28 (m, 2H), 1.96-1.80 (m, 2H), 1.69 (s, 3H), 1.62 (s, 3H), 1.33 (d, J = 7.1 Hz, 1.9H), 1.27 (d, J = 7.1 Hz, 1.1H). MS (ESI + ) C 22 H 28 INO4P + [M+H] + Calculated value: 528.08, actual value: 528.26.

[0193] (E)-((5-hydroxy-4-methylpent-3-en-1-yl)(4-iodophenoxy)phosphoryl)alanine benzyl ester (XS4) Allylic oxidation of alkene XS3 (0.434 g, 0.823 mmol) was carried out according to general procedure XXC. The crude product was purified by flash chromatography (silica gel, 40-100% EtOAc in heptane) to give alcohol XS4 (0.254 g, 57%, ca. 3:2 diastereomeric mixture) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.61-7.54 (m, 2H), 7.40-7.28 (m, 5H), 7.00-6.93 (m, 2H), 5.47-5.39 (m, 1H), 5.15-5.07 (m, 2H), 4.17-4.02 (m, 1H), 4.00 (s, 2H), 3.36 (t, J = 10.3 Hz, 0.6H), 3.27 (dd, J = 11.6, 9.6 Hz, 0.4H), 2.49-2.35 (m, 2H), 2.01-1.83 (m, 2H), 1.68 (s, 3H), 1.34 (d, J = 7.0 Hz, 1.8H), 1.25 (d, J = 7.1 Hz, 1.2H). MS (ESI + ) C 22 H 28 INO5P + [M+H] + Calculated value: 544.07, actual value: 544.32.

[0194] Linker-drug compound XC13 was synthesized from XS4 according to the following reaction scheme. [ka]

[0195] ((4-(2,5,8,11,14,17,20,23-Octaoxahexaco-25-yn-26-yl)phenoxy)((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)-L-alanine benzyl ester (XC10) To XS4 (50 mg, 0.092 mmol), 2,5,8,11,14,17,20,23-octahexacos-25-yne (34.8 mg, 0.092 mmol), bis(triphenylphosphino)palladium chloride (3.23 mg, 4.60 μmol) and Cu(I)I (1.753 mg, 9.20 μmol) was added degassed Et3N (0.2 mL, 1.44 mmol). The mixture was stirred at room temperature for 4 h. After concentration, the crude product was redissolved in DCM and concentrated again. The crude product was purified by flash chromatography (silica gel, 0-11% MeOH in DCM) to give the alcohol XC10 (67 mg, 83%) as a brown oil. MS (ESI + ) C 40 H 61 NO 13 P + [M+H] + Calculated value: 794.4, actual value: 794.6.

[0196] ((4-(2,5,8,11,14,17,20,23-Octaoxahexacos-25-yn-26-yl)phenoxy)((E)-5-((((4-((S)-2-azidopropanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)phosphoryl)-L-alanine benzyl ester (XC11) Alcohol XC10 (67 mg, 0.076 mmol) was reacted with carbamate XD5 according to general procedure XXA. Flash chromatography (silica gel, 0-8% MeOH in DCM) afforded azide XC11 (54 mg, 65%) as a light brown oil. MS (ESI + ) C 54 H 78 NO 16 P + [M+H] + Calculated value: 1097.5, actual value: 1097.8.

[0197] ((4-(2,5,8,11,14,17,20,23-Octaoxahexaco-25-yn-26-yl)phenoxy)((E)-5-((((4-((S)-2-aminopropanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)phosphoryl)-L-alanine benzyl ester (XC12) A solution of azide XC11 (54 mg, 0.049 mmol) in THF (0.473 mL) / water (0.053 mL) was purged with nitrogen gas for 15 min. Tributylphosphine (0.031 ml, 0.123 mmol) was added at room temperature and the mixture was stirred at room temperature for 5.5 h and concentrated in vacuo. Flash chromatography (silica gel, 0-15% MeOH in DCM) afforded the amine XC12 (42 mg, 80%) as a pale yellow oil. MS (ESI + ) C 54 H 80 N4O 16 P + [M+H] + Calculated value: 1071.5, actual value: 1071.9.

[0198] ((4-(2,5,8,11,14,17,20,23-Octaoxahexacos-25-yn-26-yl)phenoxy)((E)-5-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)(ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)phosphoryl)-L-alanine benzyl ester (XC13) To a cooled (0 °C) solution of amine XC12 (42 mg, 0.039 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (12.2 mg, 0.039 mmol, prepared as described in WO 2013 / 122823) in DMF (1 mL) was added HATU (17.9 mg, 0.047 mmol) and DIPEA (0.014 mL, 0.078 mmol). The resulting yellow mixture was stirred at room temperature for 1.5 h and concentrated in vacuo. The residue was dissolved in EtOAc, washed with saturated aqueous NaHCO3 / water (1:1), water and brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0-10% MeOH in DCM) to give a colorless oil. The oil was purified by preparative RP-HPLC (water / MeCN, 60:40 to 10:90 gradient, no modifiers used). Product fractions were pooled and MeCN was removed by rotary evaporation. The aqueous mixture was then lyophilized to give maleimide XC13 (21 mg, 40%) as a white solid. MS (ESI + ) C 69 H 100 NO 20 P + [M+H] + Calculated value: 1363.7, actual value: 1364.1.

[0199] Example 9 Synthesis of Linker-Drug Compound XS2 Preparation of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine 2,5-dioxopyrrolidin-1-yl ester (XD20) [ka]

[0200] (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine 2,5-dioxopyrrolidin-1-yl ester (XD20) N,N′-Dicyclohexylcarbodiimide (DCC, 459 mg, 2.222 mmol) was added to a suspension of XD19 (1.05 g, 2.22 mmol) and 1-hydroxypyrrolidine-2,5-dione (256 mg, 2.22 mmol, synthesized as described in EP 2907824) in THF (40 mL) at room temperature. After stirring for 3.5 h, the mixture was filtered and the residue was washed thoroughly with DCM. The filtrate was diluted with EtOAc and then concentrated. The white solid was suspended in a small amount of EtOAc and then filtered to give the Osu ester XD20 (612 mg, 48%) as a white solid. MS (ESI + ) C 27 H 32 N5O9 + [M+H] + Calculated value: 570.2, actual value: 570.4.

[0201] Preparation of (((E)-5-((2-((S)-2-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)acetamido)-3-phenylpropanamido)acetamido)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XS2) [ka]

[0202] (((E)-5-((2-aminoacetamido)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XS1) Step 1: Acetic acid (2-((allyloxy)carbonyl)amino)acetamide) methyl ester (0.152 g, 0.659 mmol, prepared as described in Brailsford et al. Tetrahedron, 2018, 74, 1951-1956) and pyridinium p-toluenesulfonate (PPTS; 10.6 mg, 0.042 mmol) were added to a flask under nitrogen atmosphere. Alcohol XD1 (0.110 g, 0.264 mmol, prepared as described in Kadri et al. J. Med. Chem. 2020, 63, 11258-11270) in toluene (1.3 mL) was added and the reaction mixture was stirred at 80 °C for 1 h. After cooling to room temperature, Et3N (4 drops) was added and the reaction mixture was concentrated and coevaporated with DCM (1 mL). The crude product was purified by flash chromatography (silica gel, 20-100% EtOAc in DCM) to give (E)-((5-((2-(((allyloxy)carbonyl)amino)acetamido)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl-)alanine benzyl ester (0.120 g, 68% yield) as a mixture of diastereomers. MS (ESI + ) C 29 H 39 N3O8P + [M+H] + Calculated value: 588.25, actual value: 588.42.

[0203] Step 2 A 10 mL vial was purged with nitrogen gas (3× vacuum / nitrogen gas cycles) and charged with Pd(PPh3)4 (4.2 mg, 0.0036 mmol). Alloc-protected amine (0.120 g, 0.180 mmol, prepared in step 1) in DCM (1.80 mL) was then added, followed by PhSiH3 (0.155 mL, 1.26 mmol). The reaction mixture was stirred for 1 h. The reaction mixture was diluted with DCM and purified by flash chromatography (silica gel, 5-15% MeOH in DCM) to give XS1 (63.7 mg, 70%, ca. 3:2 diastereomeric mixture) as a yellow oil. 1H NMR (400 MHz, CDCl3) ppm = 8.01 (br s, 1H), 7.46-7.22 (m, 7H), 7.22-7.16 (m, 2H), 7.16-7.09 (m, 1H), 5.48 (q, J = 7.2 Hz, 1H), 5.27-4.91 (m, 2H), 4.82-4.64 (m, 2H), 4.19-4.00 (m, 1H), 3.90 (s, 2H), 3.47 (t, J = 10.2 Hz, 1H), 3.42-3.28 (m, 2H), 2.49-2.34 (m, 2H), 2.02-1.84 (m, 4H), 1.65 (s, 3H), 1.32 (d, J = 7.0 Hz, 1.8H), 1.25 (d, J = 7.1 Hz, 1.2H). MS (ESI + ) C 25 H 35 N3O6P + [M+H] + Calculated value: 504.2, actual value: 504.4.

[0204] (((E)-5-((2-((S)-2-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)acetamido)-3-phenylpropanamido)acetamido)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XS2) To a solution of amine XS1 (25.7 mg, 0.051 mmol) in DMF (0.51 mL) was added Osu ester XD20 (31.7 mg, 0.056 mmol) followed by DIPEA (0.027 mL, 0.153 mmol). The reaction mixture was stirred at room temperature for 60 min. An additional Osu ester XD20 (5.81 mg, 10.2 μmol) was added and stirred for 40 min, after which the final Osu ester XD20 (5.81 mg, 10.2 μmol) was added followed by stirring for 30 min. The reaction mixture was concentrated, coevaporated with toluene (2 mL) and dried in vacuo. The crude product was dissolved in DCM (5 mL), filtered through a syringe filter and purified by flash chromatography (silica gel, 0-8% MeOH in DCM). The product was further purified by RP-HPLC (water / MeCN, 70:30 to 45:55 gradient, no modifiers used). Evaporation of MeCN followed by lyophilization gave XS2 (14.8 mg, 30%, approximately 3:2 diastereomeric mixture). 1H NMR (400 MHz, DMSO-d6) ppm = 8.50-8.43 (m, 1H), 8.27 (t, J = 5.8 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.06 (t, J = 5.8 Hz, 1H), 8.00 (t, J = 5.8 Hz, 1H), 7.38-7.27 (m, 7H), 7.27-7.21 (m, 4H), 7.20-7.11 (m, 4H), 6.98 (s, 2H), 5.63 (dd, J = 12.5, 10.4 Hz, 0.4H), 5.52 (dd, J = 13.3, 10.0 Hz, 0.6H), 5.44-5.36 (m, 1H), 5.13-5.02 (m, 2H), 4.55-4.46 (m, 3H), 4.00-3.90 (m, 1H), 3.77 (s, 2H), 3.74-3.69 (m, 2H), 3.66 (d, J = 5.8 Hz, 2H), 3.63-3.55 (m, 1H), 3.38-3.35 (m, 2H), 3.06 (dd, J = 13.8, 4.5 Hz, 1H), 2.81 (dd, J = 13.8, 9.8 Hz, 1H), 2.31-2.19 (m, 2H), 2.11 (t, J = 7.5 Hz, 2H), 1.88-1.75 (m, 2H), 1.57-1.53 ​​(m, 3H), 1.52-1.42 (m, 4H), 1.24-1.18 (m, 3H), 1.13 (d, J = 7.1 Hz, 2H). MS (ESI + ) C 48 H 61 N7O 12 P + [M+H] + The calculated value is 958.4 and the measured value is 958.8.

[0205] Example 10 Synthesis of Rincon compound XS7

change

[0206] (2-(Methylsulfonyl)ethyl)carbamic acid (S)-4-(2-azidopropanamide) benzyl ester (XS5) Step 1 To a solution of XD7 (1.14 g, 5.18 mmol, prepared as described in Example 1) in THF (17 mL) was added bis(4-nitrophenyl)carbonate (3.15 g, 10.4 mmol) followed by DIPEA (1.36 mL, 7.76 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the crude product was stirred in Et2O (20 mL) for 15 min and filtered. This step was repeated twice and the filtrates were combined and concentrated. Flash chromatography (silica gel, 0-50% EtOAc in heptane) afforded the corresponding carbonate (1.57 g, 79%). MS (ESI + ) C 17 H 16 N5O6 + [M+H] + Calculated value: 386.1, actual value: 386.2.

[0207] Step 2: The carbonate intermediate (0.340 g, 0.882 mmol) was dissolved in THF (4.4 mL) and 2-(methylsulfonyl)ethan-1-amine hydrochloride (0.148 g, 0.926 mmol) and TEA (0.258 mL, 1.85 mmol) were added at 0 °C. After the reaction mixture was brought to room temperature and stirred for 3 h, the reaction mixture was concentrated, dissolved in EtOAc (30 mL), washed with saturated aqueous NaHCO3 (3 × 20 mL) and brine (20 mL), dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, 0-100% EtOAc in DCM) to give carbamate XS5 (0.260 g, 80%) as a white solid. 1H NMR (400 MHz, CDCl3) ppm = 8.11 (br s, 1H), 7.57-7.51 (m, 2H), 7.33 (d, J = 8.5 Hz, 2H), 5.42 (br s, 1H), 5.07 (s, 2H), 4.24 (q, J = 7.0 Hz, 1H), 3.72 (q, J = 6.1 Hz, 2H), 3.25 (t, J = 5.9 Hz, 2H), 2.93 (s, 3H), 1.65 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 14 H 19 N5NaO5S + [M+Na] + Calculated value: 392.1, actual value: 392.1.

[0208] (((E)-5-(((((4-((S)-2-aminopropanamido)benzyl)oxy)carbonyl)(2-(methylsulfonyl)ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XS6) Step 1 To a solution of carbamate XS5 (0.186 g, 0.503 mmol) in DCM (2.5 mL) was added paraformaldehyde (18 mg, 0.60 mmol). The reaction mixture was stirred for 5 min and then TMSCl (0.070 mL, 0.55 mmol) was added. The reaction mixture was stirred for 1 h, concentrated, coevaporated with DCM (1 mL), dried in vacuum for 15 min, and dissolved in DCM (2.5 mL) to give solution A. Allyl alcohol XD1 (84 mg, 0.20 mmol) was dissolved in DCM (1.3 mL), cooled to 0° C., and a portion of solution A (1.5 mL) was added, followed by 2,6-lutidine (0.070 mL, 0.60 mmol). The reaction mixture was allowed to come to room temperature and stirred for 2 h. An additional 0.50 mL of solution A and 2,6-lutidine (0.023 mL, 0.20 mmol) were added and the mixture was stirred for 1 h. An additional 0.50 mL of solution A and 2,6-lutidine (0.023 mL, 0.20 mmol) were added and the mixture was stirred overnight. A few drops of n-BuOH were added to quench the reaction and the mixture was concentrated and co-evaporated with heptane (1 mL), toluene (1 mL) and DCM (1 mL). The crude product was purified by flash chromatography (silica gel, 0-100% EtOAc in DCM) to give the two diastereomers of (((E)-5-(((((4-((S)-2-azidopropanamido)benzyl)oxy)carbonyl)(2-(methylsulfonyl)ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (0.104 g, 65%) as a colorless oil. MS (ESI + ) C 37 H 48 NO 10 P.S. + [M+H] + Calculated value: 799.3, actual value: 799.6.

[0209] Step 2 This intermediate (60 mg, 0.075 mmol) was dissolved in THF (0.68 mL) / water (0.075 mL) and the resulting solution was purged with nitrogen gas for 15 min. Tributylphosphine (0.047 mL, 0.188 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, co-evaporated with MeCN (2× 2 mL) and dried in vacuum. The crude product was purified by flash chromatography (silica gel, 0-20% MeOH in DCM) to give the amine XS6 (27.5 mg, 47%) as a mixture of diastereomers. MS (ESI + ) C 37 H 50 N4O 10 P.S. + [M+H] + Calculated value: 773.3, actual value: 773.6.

[0210] Linker-Drug XS7 Amine XS6 (28 mg, 0.036 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (12 mg, 0.039 mmol, prepared as described in WO 2013 / 122823) were dissolved in DMF (0.36 mL) and HATU (15 mg, 0.039 mmol) and DIPEA (0.025 mL, 0.14 mmol) were added. The reaction mixture was stirred at room temperature for 40 min, concentrated and co-evaporated with toluene (2× 1 mL). The residue was dissolved in EtOAc (10 mL) and washed with saturated aqueous NaHCO3 (10 mL). The aqueous phase was back-extracted with EtOAc (2× 10 mL) and the combined organic phases were washed with water (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated. The crude product was purified by preparative RP-HPLC (MilliQ water / MeCN, 70:30 to 20:80 gradient, no modifiers used) to give after lyophilization XS7 (17.1 mg, 45%) as a mixture of diastereomers. 1H NMR (400 MHz, DMSO-d6) ppm = 9.94 (s, 1H), 8.14 (d, J = 6.9 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.38-7.27 (m, 9H), 7.20-7.10 (m, 3H), 6.99 (s, 2H), 5.62 (t, J = 11.4 Hz, 0.4H), 5.56-5.47 (m, 0.6H), 5.45-5.29 (m, 1H), 5.13-5.01 (m, 4H), 4.74 (s, 2H), 4.38 (quint, J = 7.0 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 4.04-3.89 (m, 1H), 3.81-3.71 (m, 2H), 3.71-3.63 (m, 2H), 3.42-3.33 (m, 4H), 3.05-2.91 (m, 3H), 2.35-2.20 (m, 2H), 2.20-2.07 (m, 2H), 2.03-1.90 (m, 1H), 1.89-1.71 (m, 2H), 1.59-1.44 (m, 7H), 1.30 (d, J = 7.0 Hz, 3H), 1.23-1.20 (m, 1H), 1.19-1.15 (m, 2H), 1.13 (d, J = 7.3 Hz, 2H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 52 H 70 N6O 14 PS + [M+H] + The calculated value is 1065.4 and the measured value is 1065.9.

[0211] Example 11 Synthesis of Rincon compound XS25

change

[0212] (2-(Dimethylamino)ethyl)carbamic acid (S)-4-(2-azidopropanamide) benzyl ester (XS23) The PNP carbonate of XD7 was prepared as described in the synthesis of XS5. The carbonate (0.393 g, 1.02 mmol) was dissolved in THF (5.1 mL) and N,N-dimethylethane-1,2-diamine (0.137 mL, 1.25 mmol) and TEA (0.258 mL, 1.85 mmol) were added at 0 °C. The reaction mixture was brought to room temperature and stirred for 4 h. The reaction mixture was concentrated, dissolved in EtOAc (30 mL) and washed with saturated aqueous NaHCO3 (3 × 15 mL). The combined aqueous phase was back-extracted with EtOAc (25 mL) and the combined organic phase was washed with aqueous NaOH (2 × 15 mL, 1N), brine (25 mL), dried over Na2SO4 and evaporated to give the carbamate XS23 (0.299 g, 88%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) ppm = 8.11 (br s, 1H), 7.56-7.51 (m, 2H), 7.37-7.32 (m, 2H), 5.26 (br s, 1H), 5.06 (s, 2H), 4.23 (q, J = 7.0 Hz, 1H), 3.26 (q, J = 5.6 Hz, 2H), 2.39 (t, J = 6.0 Hz, 2H), 2.21 (s, 6H), 1.64 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 15 H 23 N6O3 + [M+H] + Calculated value: 335.2, actual value: 335.3.

[0213] (((E)-5-(((((4-((S)-2-aminopropanamido)benzyl)oxy)carbonyl)(2-(dimethylamino)-ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (XS24) Step 1 To a solution of carbamate XS23 (0.160 g, 0.478 mmol) in DCM (4.8 mL) was added paraformaldehyde (20 mg, 0.67 mmol). The reaction mixture was stirred for 15 min, then TMSCl (0.094 mL, 0.74 mmol) was added. The reaction mixture was stirred for 1 h, TMSCl (0.094 mL, 0.74 mmol) was added, and stirring was continued for 2.5 h. The reaction mixture was then concentrated, coevaporated with DCM (1 mL), and dried in vacuum for 15 min. The crude intermediate was suspended in DCM (4.8 mL) and a solution of XD1 (0.493 g, 1.18 mmol) in DCM (4.8 mL) was added. The reaction mixture was stirred for 15 min, followed by the addition of 2,6-lutidine (0.167 mL, 1.44 mmol). After 20 min, MeOH (2 mL) was added and the reaction mixture was concentrated. The crude product was purified by flash chromatography (silica gel, 0-20% MeOH in DCM) to give a diastereomeric mixture of (((E)-5-(((((4-((S)-2-azidopropanamido)benzyl)oxy)carbonyl)(2-(dimethylamino)ethyl)amino)methoxy)-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine benzyl ester (0.272 g, 74%) as a pale yellow oil. 1H NMR (400 MHz, DMSO-d6) ppm = 10.34 (s, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.40-7.26 (m, 9H), 7.20-7.10 (m, 3H), 5.63 (t, J = 11.4 Hz, 0.6H), 5.53 (dd, J = 13.1, 10.2 Hz, 0.4H), 5.46-5.30 (m, 1H), 5.06 (d, J = 13.3 Hz, 4H), 4.71 (s, 2H), 4.09 (q, J = 5.3 Hz, 2H), 4.07-4.02 (m, 1H), 4.02-3.92 (m, 1H), 3.86-3.69 (m, 2H), 3.17 (d, J = 5.1 Hz, 4H), 2.73-2.59 (m, 4H), 2.33-2.16 (m, 2H), 1.87-1.72 (m, 2H), 1.59-1.49 (m, 3H), 1.49-1.40 (m, 3H), 1.28-1.15 (m, 2H), 1.13 (d, J = 7.3 Hz, 1H). MS (ESI + ) C 38 H 51 N7O8P + [M+H] + Calculated value: 764.4, actual value: 764.7.

[0214] Step 2 This intermediate (75 mg, 0.098 mmol) was dissolved in THF (0.884 mL) / water (0.098 mL) and the resulting solution was purged with nitrogen gas for 15 min. Tributylphosphine (61 μL, 0.245 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, co-evaporated with MeCN (2× 2 mL) and dried in vacuo. The crude product was purified by flash chromatography (silica gel, 0-25% MeOH in DCM) to give the two diastereomers of amine XS24 (33 mg, 45%) as yellow oils. MS (ESI + ) C 38 H 53 N5O8P + [M+H] + Calculated value: 738.4, actual value: 736.7.

[0215] Linker-Drug XS25 Amine XS24 (33 mg, 0.044 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (14 mg, 0.046 mmol, prepared as described in WO 2013 / 122823) were dissolved in DMF (0.44 mL). HATU (18 mg, 0.049 mmol) and DIPEA (31 μL, 0.18 mmol) were added and the reaction mixture was stirred at room temperature for 1 h, concentrated and coevaporated with toluene (1 mL). Water (1 mL) was added, the resulting supernatant was removed and the precipitate was washed with water. The crude product was purified by preparative RP-HPLC (MilliQ water × 0.1% TFA / MeCN, 80:20 to 30:70 gradient) to give XS25 (18 mg, 39%) as a diastereomeric mixture after lyophilization. 11H NMR (400 MHz, DMSO-d6) ppm = 9.94 (s, 1H), 9.37 (br s, 1H), 8.14 (d, J = 6.9 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.38 - 7.27 (m, 9H), 7.19 - 7.10 (m, 3H), 6.99 (s, 2H), 5.63 (t, J = 11.4 Hz, 0.7H), 5.52 (dd, J = 13.1, 10.3 Hz, 0.3H), 5.46 - 5.30 (m, 1H), 5.10 - 5.06 (m, 2H), 5.06 - 5.04 (m, 2H), 4.71 (s, 2H), 4.38 (quint, J = 7.0 Hz, 1H), 4.16 (dd, J = 8.5, 6.9 Hz, 1H), 4.06 - 3.88 (m, 1H), 3.84 - 3.73 (m, 2H), 3.64 - 3.55 (m, 2H), 3.39 - 3.36 (m, 2H), 3.29 - 3.18 (m, 2H), 2.85 - 2.70 (m, 6H), 2.31 - 2.21 (m, 2H), 2.21 - 2.08 (m, 2H), 1.95 (dq, J = 13.6, 6.8 Hz, 1H), 1.88 - 1.73 (m, 2H), 1.60 - 1.52 (m, 3H), 1.52 - 1.43 (m, 4H), 1.30 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 7.0 Hz, 3H), 1.19 - 1.11 (m, 2H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 53 H 73 N7O 12 P + [M + H] + Calculated value: 1030.5, Measured value: 1030.9.

[0216] Example 12 Synthesis of Linker - Drug Compound XS12 A Preparation of Azide XS9

Chemical Structure

[0217] (4-amino-3-fluorophenyl)methanol (XS8) To a solution of (3-fluoro-4-nitrophenyl)methanol (0.610 g, 3.56 mmol) in MeOH (8.9 mL) and THF (8.9 mL) was added zinc powder (2.33 g, 35.6 mmol) and ammonium chloride (1.91 g, 35.6 mmol). The reaction mixture was stirred at room temperature for 24 h, filtered through Celite and washed with MeOH (20 mL). The filtrate was concentrated and purified by flash chromatography (silica gel, 0-10% MeOH in DCM) to give the aniline XS8 (0.312 g, 62%) as an orange oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.01 (dd, J = 11.7, 1.9 Hz, 1H), 6.98-6.87 (m, 1H), 6.75 (dd, J = 9.0, 8.1 Hz, 1H), 4.55 (s, 2H). MS (ESI + ) C7H9FNO + [M+H] + Calculated value: 142.1, actual value: 142.1.

[0218] (S)-2-Azido-N-(2-fluoro-4-(hydroxymethyl)phenyl)propenamide (XS9) A solution of (S)-2-azidopropionic acid (0.180 g, 1.56 mmol) in MeOH (1.8 mL) was added to a solution of aniline XS8 (0.309 g, 2.19 mmol) in DCM (5.9 mL). The solution was cooled to 0 °C and EEDQ (0.774 g, 3.13 mmol) was added. After 15 min, the reaction mixture was allowed to reach room temperature and stirred overnight. The reaction mixture was concentrated and purified by flash chromatography (silica gel, 0-100% EtOAc in DCM) to give amide XS9 (0.416 g, 100%). 1H NMR (400 MHz, CDCl3) ppm = 8.42-8.29 (m, 1H), 8.26 (t, J = 8.2 Hz, 1H), 7.20-7.14 (m, 1H), 7.14-7.09 (m, 1H), 4.66 (d, J = 5.6 Hz, 2H), 4.26 (q, J = 7.1 Hz, 1H), 1.79 (t, J = 5.9 Hz, 1H), 1.66 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 10 H 12 FN4O2 + [M+H] + Calculated value: 239.1, actual value: 239.2.

[0219] B. Preparation of linker-drug compound XS12 [ka]

[0220] N-(cyclopropylmethyl)-P-(4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-azidopropanamido)-3-fluorobenzyl ester (XS10) Cyclopropylmethanamine hydrochloride (0.128 g, 1.19 mmol) was suspended in DCM (1.0 mL) and cooled to -78°C. A solution of (4-methylpent-3-en-1-yl)phosphonic acid dichloride (0.300 g, 1.19 mmol, prepared from phosphonic acid diester XD15 (0.323 g, 1.50 mmol) according to general procedure XXB) in DCM (2.0 mL) was added, followed by TEA (0.333 mL, 2.39 mmol). The reaction mixture was stirred at -78°C for 1 h, brought to room temperature and stirred for 2 h. The reaction mixture was cooled to -78°C and a solution of benzyl alcohol XS9 (0.379 g, 1.43 mmol) in DCM (4.0 mL) was added, followed by TEA (0.200 mL, 1.43 mmol). The reaction mixture was brought to room temperature and stirred for 2 h. TEA (0.100 mL, 0.717 mmol) was added and the reaction mixture was stirred for 1 h and concentrated. The crude product was partitioned between EtOAc (50 mL) and hydrochloric acid (15 mL, 1 M) and the aqueous phase was back-extracted with EtOAc (3× 15 mL). The combined organic phase was washed with brine (10 mL), dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0–100% EtOAc in DCM) to afford the two diastereomers of phosphonamidate XS10 (0.194 g, 37%) as colorless oils. 1 H NMR (400 MHz, CDCl3) ppm = 8.39 (br s, 1H), 8.28 (t, J = 8.2 Hz, 1H), 7.21-7.10 (m, 2H), 5.16-5.10 (m, 1H), 5.06-4.99 (m, 1H), 4.93-4.87 (m, 1H), 4.30-4.23 (m, 1H), 2.80-2.69 (m, 2H), 2.63-2.54 (m, 1H), 2.37-2.24 (m, 2H), 1.89-1.77 (m, 2H), 1.77-1.61 (m, 9H), 0.99-0.86 (m, 1H), 0.53-0.46 (m, 2H), 0.18-0.10 (m, 2H). MS (ESI + ) C 20 H 30 FN5O3P + [M+H]+ Calculated value: 438.2, actual value: 438.4.

[0221] N-(cyclopropylmethyl)-P-((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-azidopropanamido)-3-fluorobenzyl ester (XS11) Allylic oxidation of alkene XS10 (0.183 g, 0.418 mmol) was carried out according to general procedure XXC. The crude product was purified by flash chromatography (silica gel, 0-5% MeOH in EtOAc) to give the two diastereomers of alcohol XS11 (80 mg, 44%). 1 H NMR (400 MHz, CDCl3) ppm = 8.38 (br s, 1H), 8.28 (t, J = 8.1 Hz, 1H), 7.18 (dd, J = 11.4, 1.9 Hz, 1H), 7.15-7.09 (m, 1H), 5.49-5.40 (m, 1H), 5.07-4.99 (m, 1H), 4.94-4.86 (m, 1H), 4.27 (q, J = 7.0 Hz, 1H), 3.99 (br s, 2H), 2.75 (dt, J = 8.5, 6.8 Hz, 2H), 2.64-2.55 (m, 1H), 2.43-2.31 (m, 2H), 1.92-1.74 (m, 2H), 1.68-1.65 (m, 6H), 1.62-1.51 (m, 1H), 0.98-0.86 (m, 1H), 0.53-0.47 (m, 2H), 0.15 (q, J = 4.8 Hz, 2H). MS (ESI + ) C 20 H 30 FN5O4P + [M+H] + Calculated value: 454.2, actual value: 454.5.

[0222] Linker-Drug XS12 Step 1 The azide XS11 (74 mg, 0.16 mmol) was dissolved in THF (1.5 mL) / water (0.16 mL) and the resulting solution was purged with nitrogen gas for 15 min. Tributylphosphine (0.102 mL, 0.408 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, coevaporated with MeCN (3× 1 mL) and dried in vacuum. The crude product was purified by flash chromatography (silica gel, 0-20% MeOH in DCM) to give the two diastereomers of N-(cyclopropylmethyl)-P-((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-aminopropanamido)-3-fluorobenzyl ester (51 mg, 73%) as an oil. MS (ESI + ) C 20 H 32 FN3O4P + [M+H] + Calculated value: 428.2, actual value: 428.4.

[0223] Step 2 The intermediate amine (47 mg, 0.11 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (36 mg, 0.12 mmol, prepared as described in WO 2013 / 122823) were dissolved in DMF (1.1 mL). HATU (46 mg, 0.12 mmol) and DIPEA (0.077 mL, 0.44 mmol) were added and the reaction mixture was stirred at room temperature for 30 min. HATU (4.2 mg, 0.011 mmol) was added and the reaction mixture was stirred for 15 min, concentrated and coevaporated with toluene (1 mL). The residue was partitioned between EtOAc (10 mL) and saturated aqueous NaHCO3 (10 mL). The aqueous phase was back-extracted with EtOAc (2×10 mL) and the combined organic phase was washed with water (10 mL) / brine (5 mL) and brine (10 mL), dried over Na2SO4 and concentrated. The crude product was purified by preparative RP-HPLC (milliQ water / MeCN, 90:10 to 40:60 gradient, no modifiers used) to give after lyophilization the diastereomeric mixture of XS12 (30 mg, 38%) as a white solid. 11H NMR (400 MHz, DMSO-d6) ppm = 9.67 (s, 1H), 8.22 (d, J = 7.0 Hz, 1H), 7.86 (t, J = 8.3 Hz, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.28 (dd, J = 11.7, 1.8 Hz, 1H), 7.17 (dd, J = 8.4, 1.5 Hz, 1H), 7.00 (s, 2H), 5.40 - 5.33 (m, 1H), 4.92 - 4.78 (m, 2H), 4.73 (dt, J = 11.4, 6.9 Hz, 1H), 4.64 (t, J = 5.5 Hz, 1H), 4.52 (quint, J = 7.0 Hz, 1H), 4.22 - 4.15 (m, 1H), 3.79 - 3.73 (m, 2H), 3.37 (t, J = 7.1 Hz, 2H), 2.73 - 2.63 (m, 2H), 2.25 - 2.06 (m, 4H), 2.02 - 1.89 (m, 1H), 1.73 - 1.62 (m, 2H), 1.53 (s, 3H), 1.52 - 1.43 (m, 4H), 1.31 (d, J = 7.1 Hz, 3H), 1.23 - 1.13 (m, 2H), 0.94 - 0.86 (m, 1H), 0.84 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H), 0.42 - 0.35 (m, 2H), 0.17 - 0.11 (m, 2H). MS (ESI + ) C 35 H 52 FN5O8P + [M + H] + Calculated value: 720.4, Measured value: 720.7.

[0224] Example 13 Synthesis of Linker - Drug Compound XS17 A Preparation of Azide XS14

Chemical Structure

[0225] (4 - Amino - 2 - fluorophenyl)methanol (XS13) Step 1 4-Amino-2-fluorobenzoic acid (1.00 g, 6.45 mmol) was suspended in MeOH (3.2 mL) and cooled to 0° C. Thionyl chloride (0.706 mL, 9.67 mmol) was added dropwise and then the reaction mixture was refluxed for 1 h. MeOH (5.0 mL) was added and the reaction mixture was stirred at room temperature for 2 h. The mixture was added to saturated aqueous NaHCO3 (100 mL) and the product was extracted with EtOAc (3×75 mL). The combined organic phases were washed with brine (2×50 mL), dried over Na2SO4, concentrated, coevaporated with MeOH (10 mL) and dried in vacuum to give 4-amino-2-fluorobenzoic acid methyl ester (1.01 g, 93%) as a brown solid. 1 H NMR (400 MHz, CDCl3) ppm = 7.76 (t, J = 8.4 Hz, 1H), 6.41 (dd, J = 8.6, 2.3 Hz, 1H), 6.33 (dd, J = 12.9, 2.3 Hz, 1H), 4.15 (br s, 2H), 3.86 (s, 3H). MS (ESI + ) C8H9FNO2 + [M+H] + Calculated value: 170.1, actual value: 170.2.

[0226] Step 2 To a solution of the intermediate ester (0.486 g, 2.87 mmol) in THF (19 mL) at 0° C. was added LiAlH 4 (3.59 mL, 8.62 mmol) in THF dropwise. The reaction mixture was allowed to reach room temperature and stirred for 3 h. The reaction mixture was cooled to 0 °C and quenched by portionwise addition of a mixture of Na2SO4·10H2O (3.5 g) and Celite (3.5 g). The mixture was filtered and the residue was washed with THF (10 mL). The filtrate was concentrated in vacuo to give the benzyl alcohol XS13 (0.367 g, 91%) as an off-white solid. 1H NMR (400 MHz, CDCl3) ppm = 7.13 (t, J = 8.3 Hz, 1H), 6.45-6.40 (m, 1H), 6.40-6.35 (m, 1H), 4.61 (d, J = 5.4 Hz, 2H), 3.82-3.68 (m, 2H), 1.61 (t, J = 5.9 Hz, 1H). MS (ESI + ) C7H9FNO + [M+H] + Calculated value: 142.1, actual value: 142.1.

[0227] (S)-2-Azido-N-(3-fluoro-4-(hydroxymethyl)phenyl)propenamide (XS14) A solution of (S)-2-azidopropionic acid (0.210 g, 1.83 mmol) in MeOH (2.1 mL) was added to a solution of aniline XS13 (0.361 g, 2.55 mmol) in DCM (6.8 mL). The solution was cooled to 0 °C and EEDQ (0.902 g, 3.65 mmol) was added. After 15 min, the reaction mixture was allowed to reach room temperature and stirred overnight. The reaction mixture was concentrated and purified by flash chromatography (silica gel, 0-100% EtOAc in DCM) to give amide XS14 (0.902 g, quantitative). 1 H NMR (400 MHz, CDCl3) ppm = 8.21-8.08 (m, 1H), 7.55 (dd, J = 11.8, 2.1 Hz, 1H), 7.37 (t, J = 8.3 Hz, 1H), 7.16 (dd, J = 8.3, 2.2 Hz, 1H), 4.75-4.69 (m, 2H), 4.29-4.19 (m, 1H), 1.81-1.74 (m, 1H), 1.65 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 10 H 12 FN4O2 + [M+H] + Calculated value: 239.1, actual value: 239.2.

[0228] B. Preparation of linker-drug compound XS17 [ka]

[0229] N-(cyclopropylmethyl)-P-(4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-azidopropanamido)-2-fluorobenzyl ester (XS15) Cyclopropylmethanamine hydrochloride (0.128 g, 1.19 mmol) was suspended in DCM (1.0 mL) and cooled to -78°C. A solution of (4-methylpent-3-en-1-yl)phosphonic acid dichloride (0.300 g, 1.19 mmol, prepared from phosphonic acid diester XD15 (0.323 g, 1.50 mmol) according to general procedure XXB) in DCM (2.0 mL) was added, followed by TEA (0.333 mL, 2.39 mmol). The reaction mixture was stirred at -78°C for 1 h, brought to room temperature and stirred for 2 h. The reaction mixture was cooled to -78°C and a solution of benzyl alcohol XS14 (0.379 g, 1.43 mmol) in DCM (4.0 mL) was added, followed by TEA (0.200 mL, 1.43 mmol). The reaction mixture was brought to room temperature and stirred for 2 h. TEA (0.030 mL, 0.215 mmol) was added and the reaction mixture was stirred for 1 h and concentrated. The residue was partitioned between EtOAc (50 mL) and hydrochloric acid (15 mL, 1 M) and the aqueous phase was back-extracted with EtOAc (3× 15 mL). The combined organic phase was washed with brine (10 mL), dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0–100% EtOAc in DCM) to afford the diastereomeric mixture of phosphonamidate XS15 (0.294 g, 56%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 8.27 (s, 1H), 7.60 (dd, J = 11.8, 2.1 Hz, 1H), 7.40 (t, J = 8.3 Hz, 1H), 7.15 (dd, J = 8.3, 2.1 Hz, 1H), 5.14-5.09 (m, 1H), 5.09-4.96 (m, 2H), 4.27-4.20 (m, 1H), 2.83-2.69 (m, 2H), 2.64-2.52 (m, 1H), 2.35-2.22 (m, 2H), 1.88-1.76 (m, 2H), 1.75-1.62 (m, 9H), 1.00-0.88 (m, 1H), 0.54-0.45 (m, 2H), 0.18-0.12 (m, 2H). MS (ESI + ) C 20 H 30 FN5O3P + [M+H] + Calculated value: 438.2, actual value: 438.4.

[0230] N-(cyclopropylmethyl)-P-((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-azidopropanamido)-2-fluorobenzyl ester (XS16) Allylic oxidation of alkene XS15 (0.288 g, 0.658 mmol) was carried out according to general procedure XXC. The crude product was purified by flash chromatography (silica gel, 0-5% MeOH in EtOAc) to give XS16 (64 mg, 32%) as a diastereomeric mixture. 1H NMR (400 MHz, CDCl3) ppm = 8.25 (s, 1H), 7.60 (dd, J = 11.7, 2.1 Hz, 1H), 7.39 (t, J = 8.3 Hz, 1H), 7.15 (dd, J = 8.3, 2.1 Hz, 1H), 5.46-5.40 (m, 1H), 5.08-4.97 (m, 2H), 4.24 (q, J = 7.0 Hz, 1H), 3.98 (s, 2H), 2.81-2.71 (m, 2H), 2.68-2.52 (m, 1H), 2.41-2.30 (m, 2H), 1.91-1.71 (m, 2H), 1.68-1.63 (m, 6H), 1.59-1.52 (m, 1H), 1.01-0.86 (m, 1H), 0.54-0.45 (m, 2H), 0.18-0.13 (m, 2H). MS (ESI + ) C 20 H 30 FN5O4P + [M+H] + Calculated value: 454.2, actual value: 454.5.

[0231] Linker-Drug XS17 Step 1 The azide XS16 (61 mg, 0.14 mmol) was dissolved in THF (1.2 mL) / water (0.14 mL) and the resulting solution was purged with nitrogen gas for 15 min. Tributylphosphine (84 μL, 0.336 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, coevaporated with MeCN (3×1 mL) and dried in vacuum. The crude product was purified by flash chromatography (silica gel, 0-20% MeOH in DCM) to give the two diastereomers of N-(cyclopropylmethyl)-P-((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphonamidate 4-((S)-2-aminopropanamido)-2-fluorobenzyl ester (44 mg, 77%) as an oil. MS (ESI + ) C 20 H 32 FN3O4P + [M+H] + Calculated value: 428.2, actual value: 428.6.

[0232] Step 2 The intermediate amine (44 mg, 0.10 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (34 mg, 0.11 mmol, prepared as described in WO 2013 / 122823) were dissolved in DMF (1.0 mL). HATU (43 mg, 0.11 mmol) and DIPEA (72 μL, 0.41 mmol) were added and the reaction mixture was stirred at room temperature for 90 min, concentrated and coevaporated with toluene (1 mL). The residue was partitioned between EtOAc (10 mL) and saturated NaHCO3 solution (10 mL). The aqueous phase was back-extracted with EtOAc (2×10 mL) and the combined organic phases were washed with water (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated. The crude product was purified by preparative RP-HPLC (MilliQ water / MeCN, 90:10 to 40:60 gradient, no modifiers were used). Evaporation of MeCN followed by lyophilization afforded XS17 (32 mg, 43%) as a diastereomeric mixture. 11H NMR (400 MHz, DMSO-d6) ppm = 10.13 (s, 1H), 8.19 (d, J = 6.8 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.59 (dd, J = 12.6, 1.9 Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H), 7.31 (dd, J = 8.4, 2.0 Hz, 1H), 6.99 (s, 2H), 5.38 - 5.31 (m, 1H), 4.91 - 4.79 (m, 2H), 4.70 (dt, J = 11.4, 6.8 Hz, 1H), 4.66 - 4.60 (m, 1H), 4.36 (quint, J = 7.0 Hz, 1H), 4.16 (dd, J = 8.5, 6.9 Hz, 1H), 3.75 (d, J = 5.6 Hz, 2H), 3.36 (t, J = 7.1 Hz, 2H), 2.73 - 2.59 (m, 2H), 2.22 - 2.07 (m, 4H), 1.96 (dq, J = 13.6, 6.8 Hz, 1H), 1.70 - 1.58 (m, 2H), 1.52 (s, 3H), 1.51 - 1.42 (m, 4H), 1.30 (d, J = 7.1 Hz, 3H), 1.23 - 1.13 (m, 2H), 0.90 - 0.88 (m, 1H), 0.86 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.41 - 0.34 (m, 2H), 0.18 - 0.09 (m, 2H). MS (ESI + ) C 35 H 52 FN5O8P + [M + H] + Calculated value: 720.4, Measured value: 720.7.

[0233] Example 14 Synthesis of Linker - Drug Compound XS22 A Preparation of Azide XS19

Chemical Structure

[0234] L-Alanine 4-((S)-2-azidopropanamide) benzyl ester (XS19) Step 1 Fmoc-Ala-OH (0.386 g, 1.240 mmol) was dissolved in DCM (12 mL) and cooled to 0° C. DMAP (12 mg, 0.099 mmol) was added followed by EDC (0.291 g, 1.52 mmol) and HOBt (0.155 g, 1.01 mmol). After stirring for 15 min, alcohol XD7 (0.300 g, 1.36 mmol) was added and the reaction mixture was allowed to reach room temperature and stirred overnight. The reaction mixture was added to water (15 mL) and the aqueous phase was extracted with DCM (2×15 mL). The combined organic phase was washed with brine (15 mL), dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0-80% EtOAc in heptane) to give (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine 4-((S)-2-azidopropanamide)benzyl ester (0.406 g, 64%) as a white solid. MS (ESI + ) C 28 H 28 N5O5 + [M+H] + Calculated value: 514.2, actual value: 514.5.

[0235] Step 2: The intermediate ester (0.404 g, 0.786 mmol) was dissolved in DMF (5.3 mL) and piperidine (0.389 mL, 3.93 mmol) was added. The reaction mixture was stirred for 20 min, then concentrated and coevaporated with toluene (2× 5 mL). The crude product was purified by flash chromatography (silica gel, 0-15% MeOH in DCM) to give the amine XS19 (0.219 g, 96%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) ppm = 10.22 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 5.05 (d, J = 2.3 Hz, 2H), 4.03 (q, J = 6.9 Hz, 1H), 3.44 (q, J = 6.9 Hz, 1H), 1.79 (br s, 2H), 1.45 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 13 H 18 N5O3 + [M+H] + Calculated value: 292.1, actual value: 292.3.

[0236] B. Preparation of linker-drug compound XS22 [ka]

[0237] ((4-Methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine 4-((S)-2-azidopropanamide)benzyl ester (XS20) To (4-methylpent-3-en-1-yl)phosphonic acid dichloride (0.176 g, 0.700 mmol, prepared from phosphonic acid diester XD15 (0.189 g, 0.875 mmol) according to general procedure XXB) in toluene (4.5 mL) was added dropwise at -78 °C a solution of phenol (66 mg, 0.70 mmol) and DIPEA (0.122 mL, 0.700 mmol) in toluene (4.5 mL). The reaction mixture was allowed to reach room temperature and stirred for 2.5 h. Amine XS19 (0.214 g, 0.735 mmol) and DIPEA (0.128 mL, 0.735 mmol) were added at -78 °C and the reaction mixture was allowed to reach room temperature and stirred for 2 h. DIPEA (0.128 mL, 0.735 mmol) was added and the reaction mixture was stirred for 90 min and concentrated. The crude product was purified by flash chromatography (silica gel, 0-100% EtOAc in DCM) to afford the diastereomeric mixture of phosphonamidate XS20 (0.100 g, 28%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) ppm = 8.13 (br s, 1H), 7.57-7.51 (m, 2H), 7.37-7.27 (m, 4H), 7.22-7.08 (m, 3H), 5.17-5.08 (m, 1H), 5.07-5.01 (m, 2H), 4.24 (qd, J = 7.0, 1.6 Hz, 1H), 4.16-4.04 (m, 1H), 3.29 (t, J = 10.1 Hz, 0.5H), 3.18 (td, J = 10.4, 3.4 Hz, 0.5H), 2.40-2.30 (m, 2H), 1.96-1.81 (m, 2H), 1.77-1.73 (m, 3H), 1.69 (br s, 3H), 1.65 (d, J = 7.0 Hz, 3H), 1.25 (q, J = 7.0 Hz, 3H). MS (ESI + ) C 25 H 33 N5O5P + [M+H] + Calculated value: 514.2, actual value: 514.5.

[0238] (((E)-5-Hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine 4-((S)-2-azidopropanamide)benzyl ester (XS21) The allylic oxidation of alkene XS20 (0.100 g, 0.195 mmol) was carried out according to general procedure XXC. The crude product was purified by flash chromatography (silica gel, 0-100% EtOAc in DCM) to give the two diastereomers of alcohol XS21 (27 mg, 26%). 1 H NMR (400 MHz, CDCl3) ppm = 8.48-8.34 (m, 1H), 7.58-7.51 (m, 2H), 7.32-7.23 (m, 4H), 7.22-7.16 (m, 2H), 7.15-7.09 (m, 1H), 5.44-5.35 (m, 1H), 5.12-5.01 (m, 2H), 4.22-4.16 (m, 1H), 4.16-3.99 (m, 1H), 3.98 (s, 2H), 3.45 (t, J = 10.2 Hz, 0.5H), 3.30 (dd, J = 11.4, 10.3Hz, 0.5H), 2.49-2.32 (m, 2H), 2.00-1.80 (m, 2H), 1.65 (d, J = 6.0 Hz, 3H), 1.62 (d, J = 7.0 Hz, 3H), 1.29 (d, J = 7.1 Hz, 1.4H), 1.21 (d, J = 7.1 Hz, 1.6H). MS (ESI + ) C 25 H 33 N5O6P + [M+H] + Calculated value: 530.2, actual value: 530.5.

[0239] Linker-Drug XS22 Step 1 The azide XS21 (27 mg, 0.050 mmol) was dissolved in THF (0.45 mL) / water (0.050 mL) and the resulting solution was purged with nitrogen gas for 15 min. Tributylphosphine (0.032 mL, 0.126 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, coevaporated with MeCN (3× 1 mL) and dried in vacuum. The crude product was purified by flash chromatography (silica gel, 0-20% MeOH in DCM) to give the two diastereomers of (((E)-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alanine 4-((S)-2-aminopropanamide) benzyl ester (15 mg, 58%) as colorless oils. MS (ESI + ) C 25 H 35 N3O6P + [M+H] + Calculated value: 504.2, actual value: 504.6.

[0240] Step 2 The intermediate amine (15 mg, 0.029 mmol) and (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valine (9.9 mg, 0.032 mmol, prepared as described in WO 2013 / 122823) were dissolved in DMF (0.29 mL). HATU (13 mg, 0.035 mmol) and DIPEA (0.020 mL, 0.12 mmol) were added and the reaction mixture was stirred at room temperature for 75 min, concentrated and co-evaporated with toluene (1 mL). The residue was partitioned between EtOAc (10 mL) and saturated NaHCO3 solution (10 mL). The aqueous phase was back-extracted with EtOAc (2×10 mL) and the combined organic phases were washed with water (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated. The crude product was purified by preparative RP-HPLC (MilliQ water / MeCN, 80:20 to 30:70 gradient, no modifiers used) to give the diastereomeric mixture of linker-drug compound XS22 (10 mg, 44%) as a white solid after lyophilization. 1H NMR (400 MHz, DMSO-d6) ppm = 9.94 (s, 1H), 8.14 (d, J = 6.9 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.37-7.22 (m, 4H), 7.17-7.10 (m, 3H), 6.99 (s, 2H), 5.58 (dd, J = 12.5, 10.4 Hz, 0.5H), 5.48 (dd, J = 13.2, 10.1 Hz, 0.5H), 5.35 (t, J = 7.1 Hz, 1H), 5.07-4.94 (m, 2H), 4.65 (td, J = 5.6, 0.8 Hz, 1H), 4.38 (quint, J = 7.0 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 4.01-3.87 (m, 1H), 3.77 (d, J = 5.5 Hz, 2H), 3.36 (t, J = 7.1 Hz, 2H), 2.31-2.20 (m, 2H), 2.20-2.07 (m, 2H), 2.03-1.91 (m, 1H), 1.87-1.74 (m, 2H), 1.55-1.52 (m, 3H), 1.52-1.43 (m, 4H), 1.30 (d, J = 7.0 Hz, 3H), 1.21-1.06 (m, 5H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 40 H 55 N5O 10 P + [M+H] + The calculated value is 796.4 and the measured value is 796.6.

[0241] Example 15 Synthesis of リンカー-drug compound XD24 A Manufactured by カルバミンエステルXD26

change

[0242] Ethyl carbamic acid (S)-4-(2-(2,2,2-trifluoroacetamido)propanamide) benzyl ester (XD26) Step 1. A solution of amide XD25 (11.4 g, 61.6 mmol, prepared as described in ZP Tachrim et al. Molecules, 2007, 22, 1748) in DCM (275 mL) and MeOH (175 mL) was cooled to 0° C. and EEDQ (30.5 g, 123 mmol) and (4-aminophenyl)methanol (10.6 g, 86.0 mmol) were added. After 1 h, the orange solution was allowed to warm to room temperature and stirred overnight. The reaction was concentrated and the crude product was stirred in ether (500 mL) at room temperature for 1 h. The mixture was filtered and the solid was washed with ether to give the first crop of (S)-N-(4-(hydroxymethyl)phenyl)-2-(2,2,2-trifluoroacetamido)propanamide (10.8 g, 61%) as a white solid. The filtrate was concentrated and the crude product was dissolved in EtOAc (150 mL). The solution was washed with hydrochloric acid (2M, 90 mL) and the aqueous phase was back-extracted with EtOAc (3×125 mL). The combined organic phase was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated. The crude product was suspended in a small amount of DCM and stirred for 30 min, then the thick cake was filtered. The solid was collected and dried in vacuo to give a second crop of (S)-N-(4-(hydroxymethyl)phenyl)-2-(2,2,2-trifluoroacetamido)propanamide (4.66 g, 26% yield) as a cream solid. MS (ESI + ) C 12 H 14 F3N2O3[M+H] + Calculated value: 291.10, actual value: 291.24.

[0243] Step 2: To a solution of (S)-N-(4-(hydroxymethyl)phenyl)-2-(2,2,2-trifluoroacetamido)propanamide (3.24 g, 11.2 mmol) in THF (80 mL) was added dibutyltin dilaurate (1.66 mL, 2.79 mmol) and ethyl isocyanate (1.33 mL, 16.7 mmol) at 0 °C. The cooling bath was removed and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated onto silica gel and purified by flash chromatography (first removing the stannane impurity with a 0-80% gradient of ether in heptane, followed by eluting the product with a 0-100% gradient of EtOAc in heptane) to give the carbamate XD26 (3.62 g, 78% for two steps) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 10.18 (s, 1H), 9.71 (s, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 7.16 (br t, J = 5.4 Hz, 1H), 4.95 (s, 2H), 4.62-4.42 (m, 1H), 3.02 (qd, J = 7.2, 5.6 Hz, 2H), 1.42 (d, J = 7.3 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H). MS (ESI + ) C 15 H 18 F3N3NaO4 + [M+Na] + Calculated value: 384.1, actual value: 384.3.

[0244] B. Preparation of Linker-Drug Compound XD24 [ka]

[0245] (4-Methylpent-3-en-1-yl)phosphonic acid bis(2-cyanoethyl)ester (XD21) A solution of (4-methylpent-3-en-1-yl)phosphonic dichloride (1.04 g, 5.20 mmol, prepared from phosphonic diester XD15 according to general procedure XXB) in DCM (3.3 mL) was added dropwise to a cooled (-78 °C) solution of 3-hydroxypropanenitrile (0.746 ml, 10.9 mmol) and pyridine (0.883 ml, 10.9 mmol) in DCM (17 mL). After 30 min, the reaction was allowed to warm to room temperature. After 45 min, additional 3-hydroxypropanenitrile (0.267 ml, 3.90 mmol) and pyridine (0.315 ml, 3.90 mmol) were added at room temperature and stirring was continued for 3 h. The reaction was poured into a mixture of EtOAc (100 mL) and hydrochloric acid (1 M, 20 mL). The phases were separated and the aqueous phase was extracted with EtOAc (3 x 30 mL). The combined organic phase was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. Flash chromatography (0-100% EtOAc in DCM) afforded the dialkyl phosphonate XD21 (986 mg, 70%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) ppm = 5.12 (t, J = 7.1 Hz, 1H), 4.37-4.20 (m, 4H), 2.77 (t, J = 6.1 Hz, 4H), 2.33 (dq, J = 14.4, 7.4 Hz, 2H), 1.96-1.83 (m, 2H), 1.70 (s, 3H), 1.64 (s, 3H). MS (ESI + ) C 12 H 20 N2O3P + [M+H] + Calculated value: 271.1, actual value: 271.2.

[0246] (E)-(5-Hydroxy-4-methylpent-3-en-1-yl)phosphonic acid bis(2-cyanoethyl)ester (XD22) Allylic oxidation of alkene XD21 (0.986 g, 3.65 mmol) was carried out according to general procedure XXC. The crude product was purified by flash chromatography (silica gel, 0-6% MeOH in DCM) to give alcohol XD22 (0.586 g, 56%). 1H NMR (400 MHz, CDCl3) ppm = 5.44 (td, J = 7.1, 1.3 Hz, 1H), 4.34-4.24 (m, 4H), 4.01 (s, 2H), 2.77 (t, J = 6.0 Hz, 4H), 2.41 (dq, J = 15.5, 7.6 Hz, 2H), 2.01-1.87 (m, 2H), 1.69 (s, 3H). MS (ESI + ) C 12 H 19 N2NaO4P + [M+Na] + Calculated value: 309.1, actual value: 309.2.

[0247] (E)-(((5-(bis(2-cyanoethoxy)phosphoryl)-2-methylpent-2-en-1-yl)oxy)methyl)(ethyl)carbamic acid (S)-4-(2-(2,2,2-trifluoroacetamido)propanamide) benzyl ester (XD23) Alcohol XD22 (100 mg, 0.349 mmol) was reacted with carbamate XD26 following general procedure XXA using 2,6-lutidine instead of DIPEA. The crude product was purified by flash chromatography (silica gel, 0-4% MeOH in DCM) to give carbamate XD23 (217 mg, 94%) as a colorless oil. 1H NMR (measured at 400 MHz, DMSO-d6, 330K) ppm = 10.11 (s, 1H), 9.60 (br d, J = 6.9 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 5.40 (br s, 1H), 5.07 (s, 2H), 4.70 (s, 2H), 4.51 (quint, J = 7.1 Hz, 1H), 4.23-4.10 (m, 4H), 3.78 (br s, 2H), 3.30 (q, J = 7.0 Hz, 2H), 2.91 (t, J = 5.9 Hz, 4H), 2.26 (dq, J = 14.1, 7.2 Hz, 2H), 1.95-1.81 (m, 2H), 1.58 (br s, 3H), 1.43 (d, J = 7.1 Hz, 3H), 1.09 (t, J = 7.1 Hz, 3H). MS (ESI + ) C 28 H 38 F3N5O8P + [M+H] + Calculated value: 660.2, actual value: 660.6.

[0248] Linker-Drug XD24 Step 1 Amide XD23 (44.5 mg, 0.067 mmol) was dissolved in MeOH (1.1 mL). Water (0.135 mL) was added and the mixture was cooled to 0° C. Aqueous NaOH (2 M, 0.135 ml, 0.270 mmol) was added and after 2 min the ice bath was removed and stirring was continued at room temperature. After 2 h, more aqueous NaOH (2 M, 0.135 ml, 0.270 mmol) was added and the reaction was continued at room temperature for a total of 9 h. The reaction was cooled to 0° C. and hydrochloric acid (1 M, 0.304 mL) was added. The solution was then used in the next step without further purification.

[0249] Step 2 The solution from step 1 was treated with aqueous AcOH (1M, 0.170 mL) and the mixture was concentrated. The crude product was dissolved in water (0.4 mL) and solid NaHCO3 (16.9 mg, 0.201 mmol) was added, followed by Fmoc-Val-OSu (29.4 mg, 0.067 mmol) in THF (0.4 mL) at room temperature. The mixture was stirred at room temperature for 24 hours and then concentrated.

[0250] Step 3 The crude product prepared in step 3 was suspended in DMF (2.0 mL) and piperidine (0.8 mL) was added at room temperature. After stirring for 1 h, the reaction was concentrated and dissolved again in DMF (2.0 mL). Et3N (0.8 mL) was added and the mixture was stirred for 5 min to obtain a final suspension. The mixture was concentrated and this step was repeated again to completely remove residual piperidine. The white solid was suspended in ether (5 mL) and the mixture was stirred for 30 min. The supernatant was carefully removed and this step was repeated until no Fmoc residue was present in the supernatant by UPLC-MS analysis. The solid was dried in vacuum.

[0251] Step 4 The solid was dissolved in water (0.4 mL) and solid NaHCO3 (17.0 mg, 0.200 mmol) was added followed by 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (20.8 mg, 0.068 mmol) in THF (0.4 mL) at room temperature. After stirring for 3 h, the majority of the THF was removed by brief rotary evaporation at room temperature. The aqueous solution was then diluted with 10% MeCN in Milli-Q water (8 mL) and the clear solution was purified by preparative RP-HPLC (water × 0.025% NH4OH / MeCN, 10-40% gradient). Note that to ensure direct acidification of the basic eluate, the product was collected in a test tube previously containing aqueous AcOH (1 M, 0.5 mL). The product fractions were immediately frozen and then lyophilized to give the title compound XD24 (11.5 mg) as a white solid. 1H NMR (400 MHz, DO) ppm = 7.49-7.39 (m, 2H), 7.39-7.30 (m, 2H), 6.73 (s, 2H), 5.53-5.30 (m, 1H), 5.09 (br s, 2H), 4.70 (s, 2H; hidden by residual solvent peaks in DO, according to correlations observed by HSQC), 4.39 (q, J = 6.8 Hz, 1H), 4.03 (br d, J = 7.4 Hz, 1H), 3.87-3.72 (m, 2H), 3.36 (t, J = 6.8 Hz, 2H), 3.34-3.26 (m, 2H), 2.24 (t, J = 6.8 Hz, 2H), 2.20-2.08 (m, J = 4.1 Hz, 2H), 2.01 (dq, J = 13.6, 6.8 Hz, 1H), 1.64-1.37 (m, 12H), 1.24-1.11 (m, 2H), 1.06 (t, J = 7.1 Hz, 3H), 0.96-0.81 (m, 6H). MS (ESI - ) C 35 H 51 N5O 11 P - [MH] - Calculated value: 748.3, actual value: 748.8.

[0252] Example 17 Synthesis of Linker-Drug Compounds XD44, XD45, and XD46 A. Preparation of bis((9H-fluoren-9-yl)methyl)phosphochloridate (XD34) [ka]

[0253] Bis((9H-fluoren-9-yl)methyl)phosphonate (XD50) (9H-Fluoren-9-yl)methanol (4.55 g, 23.2 mmol) was added to a solution of diphenyl phosphonate (2.13 ml, 10.6 mmol) in dry pyridine (20 mL) at room temperature under nitrogen atmosphere and the mixture was stirred for 2 h. The reaction was concentrated and dissolved in EtOAc (250 mL). The organic phase was washed with hydrochloric acid (2×, 1 M) and brine, dried over Na2SO4, filtered, and concentrated onto silica gel. Flash chromatography (silica gel, 0-85% EtOAc / DCM (1:4) in heptane) afforded the phosphonic acid XD50 (3.46 g, 75%) as a colorless wax. 1 H NMR (400 MHz, CDCl3) ppm = 7.76-7.66 (m, 4H), 7.58-7.45 (m, 4H), 7.42-7.31 (m, 4H), 7.31-7.22 (m, 4H), 7.19-7.12 (m, 1H), 6.68 (d, J = 705.8 Hz, 1H), 4.34-4.21 (m, 4H), 4.15-4.08 (m, 2H). MS (ESI + ) C 28 H 24 O3P + [M+H] + Calculated value: 439.2, actual value: 439.3.

[0254] Bis((9H-fluoren-9-yl)methyl)phosphoric acid chloride (XD34) Phosphonic acid XD50 (8.57 g, 19.6 mmol) was dissolved in toluene (98 mL) and the overhead space was purged with nitrogen gas. NCS (3.13 g, 23.5 mmol) was added at room temperature and the reaction mixture was then stirred at 40 °C for 2 h. After cooling to room temperature, the reaction mixture was filtered and concentrated. The residue was co-evaporated with MeCN (10 mL) to give a white solid. All the solid was dissolved in MeCN (25 mL) by gentle heating with a heat gun. The solution was gradually cooled to -30 °C and a white solid started to precipitate. The flask was kept at -30 °C overnight and then warmed to room temperature before filtering. The solid was washed with ice-cold MeCN (10 mL) to give chloride XD34 (8.03 g, 87% yield) as a white solid. 1H NMR (400 MHz, CDCl3) ppm = 7.76-7.71 (m, 4H), 7.56-7.48 (m, 4H), 7.43-7.36 (m, 4H), 7.33-7.25 (m, 4H), 4.46 (dt, J = 9.7, 7.1 Hz, 2H), 4.36-4.28 (m, 2H), 4.25-4.19 (m, 2H). MS (ESI + ) C 28 H 24 ClO4P + [M+NH4] + Calculated value: 490.1, actual value: 490.3.

[0255] B. Preparation of Phosphonate Ester XD37 [ka]

[0256] (E)-(5-Hydroxy-4-methylpent-3-en-1-yl)phosphonic acid triethylammonium salt (XD37) DBU (0.401 ml, 2.66 mmol) was added to a solution of XD22 (305 mg, 1.07 mmol) in THF (10 mL) at 0 °C. The cooling bath was removed after 2 min and the reaction was stirred at room temperature for 4 h, giving exclusively one deprotected product. The mixture was diluted with ether (30 mL) and stirred for 15 min, after which the emulsion was allowed to settle (15 min). The supernatant was decanted and the residue was dissolved in MeOH (10 mL) and water (1.25 mL). Aqueous NaOH (2 M, 3.20 mL, 6.40 mmol) was added at 0 °C and the mixture was stirred at this temperature for 30 min. The ice bath was removed and the reaction was continued at room temperature for 3.5 h. The reaction mixture was then loaded onto a DOWEX 50WX8-200 hydrogen column (5 g, prewashed with methanol until the eluate was colorless and neutral). The product was eluted with methanol. The product fractions were pooled and EtN (0.148 mL, 1.07 mmol) was added. The methanol was removed by rotary evaporation and the aqueous phase was diluted with MeCN and then lyophilized to give XD37 (259 mg, 98%) as a colorless oil.1 H NMR (400 MHz, CD3OD) ppm = 5.51-5.42 (m, 1H), 3.91 (s, 2H), 3.19 (q, J = 7.3 Hz, 6H), 2.40-2.27 (m, 2H), 1.67 (s, 3H), 1.69-1.58 (m, 2H), 1.31 (t, J = 7.3 Hz, 9H). MS (ESI - ) C6H 12 O4P - [MH] - Calculated value: 179.1, actual value: 179.1.

[0257] C. Preparation of phosphate ester XD38 [ka]

[0258] (E)-Bis((9H-fluoren-9-yl)methyl) (4-((tert-butyldiphenylsilyl)oxy)-3-methylbut-2-en-1-yl) phosphate ester (XD48) To a solution of alcohol XD47 (1.35 g, 3.97 mmol, synthesized as described in Serra, S. Tetrahedron: Asymmetry, 2014, 25, 1561-1572), 5-(ethylthio)-1H-tetrazole (0.043 g, 0.331 mmol) and 2,6-lutidine (1.54 ml, 13.2 mmol) in MeCN (5 mL) at 0 °C was added XD34 (1.45 g, 3.31 mmol) in MeCN / DCM (1:1, 5 mL). The cooling bath was removed and the reaction was stirred at room temperature for 75 min. The mixture was concentrated and the crude product was dissolved in EtOAc (100 mL). The suspension was washed with hydrochloric acid (1 M, ca. 50 mL) and the aqueous phase was back extracted with EtOAc (1×). The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography (silica gel, 0-60% Et2O / DCM (1:1) in heptane) to give XD48 (1.95 g, 76%). 1H NMR (400 MHz, CDCl3) ppm = 7.76-7.69 (m, 4H), 7.67-7.61 (m, 4H), 7.58-7.49 (m, 4H), 7.45-7.31 (m, 10H), 7.30-7.22 (m, 4H), 5.75-5.67 (m, 1H), 4.54 (t, J = 7.6 Hz, 2H), 4.30-4.23 (m, 4H), 4.20-4.13 (m, 2H), 4.02 (s, 2H), 1.56 (s, 3H), 1.04 (s, 9H). MS (ESI + ) C 49 H 49 NaO5PSi + [M+H] + Calculated value: 799.3, actual value: 799.6.

[0259] (E)-4-Hydroxy-3-methylbut-2-en-1-yl triethylammonium hydrogen phosphate (XD38) Step 1 TBDPS-ether XD48 (911 mg, 1.17 mmol) was dissolved in THF / pyridine (1:1, 6 mL) in a Teflon tube under nitrogen atmosphere. The mixture was cooled to 0 °C and HF·py (2 mL, 70% HF) was added. After stirring at 0 °C for 80 min, the reaction mixture was carefully poured into a cooled (0 °C) mixture of saturated aqueous NaHCO3 and EtOAc with stirring. After effervescence ceased, the phases were separated and the aqueous phase was extracted with EtOAc (3x). The combined organic phase was washed with hydrochloric acid (1 M) and brine, dried over Na2SO4, filtered and concentrated. Flash chromatography (silica gel, 0-55% EtOAc in DCM) afforded the corresponding alcohol (418 mg, 66%).

[0260] Step 2 Alcohol (313 mg, 0.581 mmol) was dissolved in THF (13 mL). The mixture was cooled to -78°C and DBU (0.219 ml, 1.45 mmol) was added. After 5 min the cooling bath was removed and the mixture was stirred at room temperature for 45 min. A sticky precipitate formed during this time. The reaction was diluted with ether (~10 mL) and after stirring for 2 min the reaction was decanted. The residue was dissolved in a small amount of MeCN (~2 mL) and a small amount of MeOH was added to obtain a clear solution. The reaction was then diluted with ether (~70 mL). The cloudy mixture was stirred for 5 min and then allowed to stand overnight. The supernatant was decanted and then the process of dissolving in MeCN / MeOH and precipitating with ether was repeated three times. The residue was dried in vacuum to give 209 mg of an oil. This material was dissolved in ethanol (2 mL) and loaded onto a DOWEX 50WX8-200 hydrogen column (5 g, pre-washed with ethanol until the eluent was colorless and neutral). The product was eluted with ethanol. The product fractions were combined, Et3N (0.083 mL) was added, and the clear, colorless solution was concentrated to give XD38 (125 mg, 76%) as a colorless oil. 1 H NMR (400 MHz, CD3OD) ppm = 5.71-5.59 (m, 1H), 4.47 (t, J = 6.8 Hz, 2H), 3.95 (s, 2H), 3.25-3.11 (m, 6H), 1.70 (s, 3H), 1.31 (t, J = 7.3 Hz, 9H). MS (ESI - ) C5H 10 O5P - [MH] - Calculated value: 181.0, actual value: 181.1.

[0261] D. Preparation of Thiophosphate Ester XD39 [ka]

[0262] (E)-tert-Butyl((4-chloro-2-methylbut-2-en-1-yl)oxy)diphenylsilane (XD51) NCS (1.02 g, 7.63 mmol) was dissolved in dry DCM (25 mL) and the mixture was cooled to -40 °C. DMS (0.695 ml, 9.40 mmol) was added dropwise with stirring, then the reaction was warmed to 0 °C and stirred for 10 min. The reaction was cooled to -40 °C and alcohol XD47 (2.00 g, 5.87 mmol, synthesized as described in Serra, S. Tetrahedron: Asymmetry, 2014, 25, 1561-1572) dissolved in dry DCM (5 mL) was added. The reaction was warmed to 0 °C over 2.5 h and then stirred at 0 °C for an additional 90 min. Brine (30 mL) was added at 0 °C and the phases were separated. The aqueous phase was extracted with DCM (40 mL) and the combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The crude nearly colorless oil was purified by flash chromatography (silica gel, 0-20% DCM in heptane) to give chloride XD51 (1.93 g, 92%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.72-7.64 (m, 4H), 7.47-7.36 (m, 6H), 5.85 (tq, J = 8.1, 1.5 Hz, 1H), 4.16 (d, J = 8.1 Hz, 2H), 4.09 (s, 2H), 1.68 (s, 3H), 1.08 (s, 9H).

[0263] Thiophosphoric acid (E)-O,O-bis((9H-fluoren-9-yl)methyl) S-(4-((tert-butyldiphenylsilyl)oxy)-3-methylbut-2-en-1-yl) ester (XD52) Step 1 Sodium methanethiosulfonate (262 mg, 1.95 mmol) was added to a solution of chloride XD51 (700 mg, 1.95 mmol) in DMF (4 mL) at room temperature. After stirring for 5 h, the mixture was poured into water (50 mL), the mixture was extracted with EtOAc / heptane (1:1, 2× 30 mL), and the combined organic phases were washed with water (2× 30 mL) and brine (30 mL), dried over Na2SO4, filtered, and concentrated. Flash chromatography (silica gel, 0-15% EtOAc in heptane) afforded methanethiosulfonic acid (E)-S-(4-((tert-butyldiphenylsilyl)oxy)-3-methylbut-2-en-1-yl) ester (725 mg, 86%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71-7.61 (m, 4H), 7.51-7.34 (m, 6H), 5.75 (tq, J = 7.9, 1.4 Hz, 1H), 4.10 (s, 2H), 3.91 (d, J = 7.9 Hz, 2H), 3.27 (s, 3H), 1.70 (s, 3H), 1.08 (s, 9H).

[0264] Step 2 XD50 (570 mg, 1.30 mmol) was dissolved in MeCN (2.75 mL) and pyridine (5.6 mL) under nitrogen atmosphere. The solution was cooled on an ice bath and TMSCl (0.825 ml, 6.51 mmol) was added dropwise. After 5 min the cooling bath was removed and the reaction was stirred at room temperature for 45 min. Methanethiosulfonic acid (E)-S-(4-((tert-butyldiphenylsilyl)oxy)-3-methylbut-2-en-1-yl) ester (706 mg, 1.62 mmol) was then added and the mixture was stirred at room temperature for 15 min. Toluene (10 mL) was added and the mixture was concentrated. Again the residue was co-evaporated with toluene (10 mL) and purified by flash chromatography (silica gel, 0-50% "1:1 ether / DCM" in heptane) to give XD52 (818 mg, 79%) as a colorless wax. 1H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 6.9 Hz, 4H), 7.65-7.59 (m, 4H), 7.59-7.52 (m, 4H), 7.44-7.32 (m, 10H), 7.32-7.24 (m, 4H), 5.63-5.50 (m, 1H), 4.51-4.36 (m, 2H), 4.33-4.15 (m, 4H), 3.96 (s, 2H), 3.40 (dd, J = 12.0, 8.0 Hz, 2H), 1.53 (s, 3H), 1.02 (s, 9H).

[0265] Thiophosphate O-hydrogen (E)-S-(4-hydroxy-3-methylbut-2-en-1-yl) ester triethylammonium (XD39) Step 1 TBDPS-ether XD52 (800 mg, 1.01 mmol) was reacted with HF·py for 1 h 45 min as described in the procedure for XD38. The crude product was purified by flash chromatography (silica gel, 0–40% EtOAc in DCM) to give the corresponding alcohol (452 ​​mg, 81%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.75 (t, J = 6.8 Hz, 4H), 7.61-7.53 (m, 4H), 7.45-7.36 (m, 4H), 7.36-7.27 (m, 4H), 5.45 (tq, J = 8.0, 1.4 Hz, 1H), 4.48-4.39 (m, 2H), 4.31-4.19 (m, 4H), 3.89 (d, J = 6.1 Hz, 2H), 3.36 (dd, J = 13.6, 7.9 Hz, 2H), 1.59 (s, 3H). MS (ESI + ) C 33 H 32 O4PS + [M+H] + Calculated value: 555.2, actual value: 555.5.

[0266] Step 2 The alcohol (272 mg, 0.490 mmol) was dissolved in THF (3 mL) and Et3N (0.75 ml, 5.38 mmol) was added at room temperature. After 7 h an oily precipitate formed and MeCN (2 mL) was added followed by triethylamine (0.5 ml, 3.59 mmol) to give a clear solution. Stirring was continued overnight and then the clear solution was concentrated to about 1 mL and coevaporated with toluene (6 mL). The oily residue was dissolved in MeCN (about 0.7 mL) and then precipitated by slowly adding ether (7 mL) with stirring. After stirring for 5 min, the emulsion was allowed to settle and the solution was then decanted off leaving an oily material. The MeCN / ether treatment was repeated four times and the residue was then dried in vacuum to give the triethylamine salt XD39 (123 mg, 83%) as a colorless oil. 1 H NMR (400 MHz, CD3OD) ppm = 5.63 (tq, J = 7.9, 1.3 Hz, 1H), 3.93 (s, 2H), 3.48 (dd, J = 9.4, 8.4 Hz, 2H), 3.19 (q, J = 7.4 Hz, 6H), 1.71 (d, J = 0.8 Hz, 3H), 1.32 (t, J = 7.3 Hz, 9H). MS (ESI - ) C5H 10 O4PS - [MH] - Calculated value: 197.0, actual value: 197.0.

[0267] E. Preparation of alkyne linker XD43 [ka]

[0268] Prop-2-yn-1-ylcarbamic acid 2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl ester (XD43) To the PNP carbonate XD53 (511 mg, 1.46 mmol, synthesized according to Elgersma, RC et al. Mol. Pharm. 2015, 12, 1813-1835) in THF (10 mL) was added propargylamine (0.093 ml, 1.46 mmol) at 0 °C. The cooling bath was removed and the mixture was stirred at room temperature for 2 h. The mixture was concentrated and the crude product was purified by flash chromatography (silica gel, 0-70% EtOAc in heptane) to give XD43 (265 mg, 68%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.60 (br t, J = 5.5 Hz, 1H), 7.02 (s, 2H), 4.07-3.96 (m, 2H), 3.74 (dd, J = 5.8, 2.4 Hz, 2H), 3.61-3.48 (m, 7H), 3.07 (t, J = 2.5 Hz, 1H).

[0269] F. General Procedure XXD: CDI Activation of Phosphate Esters and Coupling with Phosphonates, Phosphates, or Thiophosphates The monotriethylamine salt of phosphoric acid (1.0 equiv.) was dissolved in DMF (0.15 M) under nitrogen atmosphere and CDI (2.1 equiv.) was added at room temperature. After stirring for 30 min, dry MeOH (1.0 equiv.) was added and the mixture was stirred at room temperature for 15 min and concentrated. The residue was co-evaporated with DMF to give crude product A.

[0270] In a separate flask, the monotriethylamine salt of the phosphonic, phosphoric or thiophosphoric reactant (1.2 equiv.) was coevaporated with DMF and then redissolved in DMF (0.36 M) under nitrogen. The mixture was then cannulated into the flask containing crude product A (at room temperature). An equal volume of DMF was used to rinse the flask to complete the transfer. The mixture was stirred at room temperature under nitrogen until analysis by UPLC-MS showed essentially complete conversion (usually 20-24 h), at which point the reaction was concentrated and purified by preparative HPLC. Lyophilization of the product fractions afforded the product.

[0271] G. General Procedure XXE: Click Reaction Copper(II) sulfate pentahydrate (0.77 equiv.) in nitrogen-purged water (0.034 M) was added to a flask containing solid azide (1.0 equiv.) and alkyne (1.4 equiv.) at room temperature. An equal volume of THF was added to obtain a homogeneous water / THF (1:1) solution. The headspace of the flask was briefly purged with nitrogen gas, and sodium ascorbate (1.5 equiv.) in nitrogen-purged water (0.13 M) was added. The reaction was stirred at room temperature until UPLC-MS analysis showed complete conversion (usually 1-2 h). The bulk of the THF was removed by brief rotary evaporation at room temperature, and the aqueous phase was dissolved in MeCN / 25 mM NH4HCO3 (1:9) in Milli-Q water. Insoluble material was removed using a syringe filter, and the filtrate was purified by preparative HPLC. The product was obtained by lyophilization of the product fraction.

[0272] H Preparation of linker-drug compounds XD44, XD45 and XD46 [ka]

[0273] (4-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)benzyl)phosphate bis((9H-fluoren-9-yl)methyl)ester (XD35) Step 1 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propionic acid (142 mg, 0.574 mmol) was dissolved in DMF (1 mL). Val-Ala-PAB (160 mg, 0.545 mmol) in DMF (3.0 mL) was added followed by HATU (228 mg, 0.600 mmol) and DIPEA (0.143 ml, 0.818 mmol) at room temperature. The reaction was stirred for 30 min and concentrated. Basic impurities were removed by dissolving the crude product in MeOH (1 mL) and passing the solution through a short DOWEX 50WX8 plug that had been pre-washed with methanol. The product was eluted with methanol and the crude product was concentrated onto silica gel. Flash chromatography (silica gel, 0-8% MeOH in DCM) afforded the amide (262 mg, 92%) as a cream solid. MS (ESI + ) C 24 H 39 N6O7 + [M+H] + Calculated value: 523.3, actual value: 523.6.

[0274] Step 2: To the resulting amide (977 mg, 1.87 mmol) and 5-(ethylthio)-1H-tetrazole (19 mg, 0.15 mmol) in MeCN (3.7 mL) was added 2,6-lutidine (719 μl, 6.17 mmol) at room temperature under nitrogen atmosphere, followed by a solution of chloride XD34 (884 mg, 1.87 mmol) in DCM (3.7 mL) and the mixture was stirred at room temperature. Further additions of chloride XD34 were made after 80 min (88 mg, 0.187 mmol) and 140 min (177 mg, 0.374 mmol). After 185 min of reaction time, further 2,6-lutidine (218 μl, 1.87 mmol) was added and the reaction was continued for 2 h before being quenched with methanol (1 mL). The mixture was concentrated and the crude product was dissolved in EtOAc (80 mL) and hydrochloric acid (40 mL, 1 M). A small amount of MeCN (4 mL) was added to dissolve the remaining solid and the phases were separated. The aqueous phase was extracted with EtOAc (80 mL) and the combined organic phases were washed with brine and dried over Na2SO4. The crude product was purified by flash chromatography (silica gel, 0-5% MeOH in DCM) to give the phosphate ester XD35 (1.40 g, 66% yield). 1 H NMR (400 MHz, DMSO-d6) ppm = 9.94 (s, 1H), 8.18 (d, J = 7.0 Hz, 1H), 7.89-7.82 (m, 5H), 7.55 (d, J = 8.6 Hz, 2H), 7.52-7.44 (m, 4H), 7.42-7.34 (m, 4H), 7.30-7.24 (m, 4H), 7.09 (d, J = 8.6 Hz, 2H), 4.60 (d, J = 8.8 Hz, 2H), 4.40 (quint, J = 7.0 Hz, 1H), 4.25-4.17 (m, 5H), 4.15-4.11 (m, 2H), 3.62-3.56 (m, 4H), 3.55-3.46 (m, 8H), 3.39-3.36 (m, 2H), 2.50-2.36 (m, 2H), 2.02-1.93 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H). MS (ESI+ ) C 52 H 59 NO 10 PNa + [M+H] + Calculated value: 981.4, actual value: 981.8.

[0275] 4-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)benzyl phosphate (XD36) Triethylamine (0.25 mL) was added to a solution of the phosphate ester XD35 (160 mg, 0.167 mmol) in MeCN (1 mL) at room temperature and the reaction was stirred for 24 h. The reaction was diluted with toluene (8 mL) and then concentrated. The crude product was suspended in ether (10 mL), filtered, and the solid was washed repeatedly with ether to give the phosphate alkyl ester XD36 (108 mg, 92%) as the monotriethylammonium salt. (Note: this product contained an impurity (m / z 606) that may have formed from elimination of the alcohol from the phosphate ester and capture of the intermediate azaquinone methide with triethylamine. This impurity does not react in the next step and therefore no further purification is necessary.) MS (ESI - ) C 24 H 38 NO 10 P - [MH] - Calculated value: 601.2, actual value: 601.7.

[0276] Azide XD40 The phosphate alkyl ester XD36 (107 mg, 0.152 mmol) was reacted with the phosphonate ester XD37 according to general procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give the phosphonophosphate XD40 (60.5 mg, 50%) as a fluffy white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.44-7.39 (m, 4H), 5.38 (br t, J = 7.1 Hz, 1H), 4.91 (d, J = 7.0 Hz, 2H), 4.40 (q, J = 7.1 Hz, 1H), 4.12 (d, J = 7.1 Hz, 1H), 3.88 (s, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.66-3.55 (m, 10H), 3.42 (t, J = 4.9 Hz, 2H), 2.63-2.48 (m, 2H), 2.26-2.14 (m, 2H), 2.11-1.99 (m, 1H), 1.74-1.61 (m, 2H), 1.56 (s, 3H), 1.43 (d, J = 7.3 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H). MS (ESI - ) C 30 H 49 NO 13 P2 - [MH] - Calculated value: 763.3, actual value: 763.7.

[0277] Azide XD41 Phosphate alkyl ester XD36 (142 mg, 0.202 mmol) was reacted with phosphate alkyl ester XD38 according to general procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in MilliQ water, 90:10 to 50:50 gradient) to give pyrophosphate ester XD41 (65.9 mg, 41%) as a fluffy white solid after lyophilization. MS (ESI - ) C 29 H 47 NO 14 P2 - [MH] - Calculated value: 765.3, actual value: 765.6.

[0278] Azide XD42 Phosphate alkyl ester XD36 (142 mg, 0.202 mmol) was reacted with phosphate alkyl ester XD39 according to general procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give the azide XD42 (88.6 mg, 54%) as a fluffy white solid after lyophilization. 1 H NMR (400 MHz, D2O) ppm = 7.48-7.39 (m, 4H), 5.51 (td, J = 7.9, 1.1 Hz, 1H), 4.96 (d, J = 6.9 Hz, 2H), 4.40 (q, J = 7.2 Hz, 1H), 4.12 (d, J = 7.1 Hz, 1H), 3.88 (s, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.67-3.54 (m, 10H), 3.47-3.36 (m, 4H), 2.63-2.48 (m, 2H), 2.13-1.98 (m, 1H), 1.59 (s, 3H), 1.43 (d, J = 7.1 Hz, 3H), 0.92 (br d, J = 6.8 Hz, 3H), 0.91 (br d, J = 6.6 Hz, 3H). MS (ESI - ) C 29 H 47 NO 13 P2S - [MH] - Calculated value: 781.3, actual value: 781.5.

[0279] Linker-Drug XD44 The azide XD40 (18.5 mg, 0.023 mmol) was reacted with alkyne XD43 according to general procedure XXE. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the phosphonophosphate XD44 (11.6 mg, 47%) as a fluffy white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.88 (s, 1H), 7.41 (s, 4H), 6.74 (s, 2H), 5.38 (br t, J = 7.2 Hz, 1H), 4.91 (d, J = 6.8 Hz, 2H), 4.53 (t, J = 4.9 Hz, 2H), 4.38 (q, J = 7.2 Hz, 1H), 4.31 (s, 2H), 4.17-4.04 (m, 3H), 3.92-3.83 (m, 4H), 3.70 (t, J = 6.0 Hz, 2H), 3.66-3.57 (m, 6H), 3.57-3.43 (m, 8H), 2.62-2.47 (m, 2H), 2.27-2.14 (m, 2H), 2.10-1.97 (m, 1H), 1.74-1.61 (m, 2H), 1.55 (s, 3H), 1.41 (d, J = 7.3 Hz, 3H), 0.90 (d, J = 7.6 Hz, 3H), 0.88 (d, J = 7.6 Hz, 3H). MS (ESI - ) C 42 H 63 N8O 18 P2 - [MH] - Calculated value: 1029.4, actual value: 1029.8.

[0280] Linker-Drug XD45 The azide XD41 (30.2 mg, 0.038 mmol) was reacted with the alkyne XD43 according to general procedure XXE. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in MilliQ water, 90:10 to 50:50 gradient) afforded the pyrophosphate XD45 (36.2 mg, 90%) as a fluffy white solid after lyophilization. MS (ESI - ) C 41 H 61 N8O 19 P2 - [MH] - Calculated value: 1031.4, actual value: 1031.7.

[0281] Linker-Drug XD46 The azide XD42 (27.1 mg, 0.033 mmol) was reacted with the alkyne XD43 according to general procedure XXE. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in MilliQ water, 90:10 to 50:50 gradient) afforded the linker-drug compound XD46 (22.6 mg, 63%) as a fluffy white solid after lyophilization. MS (ESI - ) C 41 H 61 N8O 18 P2S - [MH] - Calculated value: 1047.3, actual value: 1047.7.

[0282] Example 18 Synthesis of Linker-Drug Compound XD58 A. Preparation of azide XD57 [ka]

[0283] (4-((S)-2-(2,2,2-trifluoroacetamido)propanamido)benzyl) (4-methylpent-3-en-1-yl)phosphonic acid 2-cyanoethyl (XD54) A solution of (4-methylpent-3-en-1-yl)phosphonic dichloride (540 mg, 2.69 mmol, prepared from phosphonic diester XD15 according to general procedure XXB) in DCM (4.3 mL) was added to a nitrogen purged vial containing 5-(ethylthio)-1H-tetrazole (35.0 mg, 0.269 mmol). The solution was cooled to -78 °C and 3-hydroxypropanenitrile (0.184 ml, 2.69 mmol) and 2,6-lutidine (0.313 ml, 2.69 mmol) were added sequentially. After stirring at -78 °C for 30 min, the reaction was allowed to warm to room temperature and stirred for 2.5 h. A solution of XD49 (780 mg, 2.69 mmol, prepared as described in the synthesis of XD26) in THF / DCM (11 mL, 3:1, gentle heating with a heat gun was required to obtain a clear solution, followed by cooling to room temperature) was then rapidly added to the reaction mixture at room temperature. After 3 h, 2,6-lutidine (0.313 ml, 2.69 mmol) was further added and the reaction continued for 90 min. The mixture was diluted with EtOAc (50 mL) and washed with hydrochloric acid (50 mL, 1 M). The aqueous phase was back-extracted with EtOAc (2× 25 mL) and the combined organic phase was washed with brine (25 mL), dried over Na2SO4, filtered and concentrated. The incompletely separated and impure product was purified again by flash chromatography (silica gel, 0–40% acetone in DCM) to give pure XD54 (630 mg, 48%). MS (ESI + ) C 21 H 27 F3N3NaO5P + [M+Na] + Calculated value: 512.2, actual value: 512.5.

[0284] (4-((S)-2-(2,2,2-trifluoroacetamido)propanamido)benzyl) ((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphonic acid 2-cyanoethyl ester (XD55) Allylic oxidation of alkene XD54 (0.625 g, 1.28 mmol) was carried out according to general procedure XXC. The crude product was purified by flash chromatography (silica gel, 0-8% MeOH in DCM) to give alcohol XD55 (0.411 g, 64%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.22 (s, 1H), 9.73 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 5.39-5.29 (m, 1H), 5.05-4.91 (m, 2H), 4.66 (t, J = 5.6 Hz, 1H), 4.48 (q, J = 7.1 Hz, 1H), 4.18-4.02 (m, 2H), 3.76 (d, J = 5.3 Hz, 2H), 2.88 (t, J = 5.9 Hz, 2H), 2.26-2.13 (m, 2H), 1.90-1.76 (m, 2H), 1.52 (s, 3H), 1.41 (d, J = 7.3 Hz, 3H). MS (ESI + ) C 21 H 27 F3N3NaO6P + [M+Na] + Calculated value: 528.2, actual value: 528.4.

[0285] Azide XD57 Step 1 Aqueous NaOH (2M, 1.31 ml, 2.62 mmol) was added to a solution of XD55 (396 mg, 0.655 mmol) in MeOH (4.6 mL) / water (0.6 mL) at 0° C. After 10 min, an additional portion of aqueous NaOH (2M, 1.31 ml, 2.62 mmol) was added and then the cooling bath was removed. After 2 h, the reaction was cooled to 0° C. and hydrochloric acid (1M, 2.95 mL, 4.5 equiv.) was added, followed by aqueous AcOH (1M, 1.97 mL, 3.0 equiv.). The mixture was then concentrated. MS (ESI + ) C 16 H 26 N2O5P + [M+H] + Calculated value: 357.2, actual value: 357.4.

[0286] Step 2 The crude product was dissolved in water (5 mL) and NaHCO3 (165 mg, 1.97 mmol) and iPrOH (5 mL) were added. Fmoc-Val-OSu (286 mg, 0.655 mmol) was added with stirring at room temperature, followed by THF (2.5 mL). After 2.5 h, the reaction was quenched with aqueous AcOH (1 M, 2 mL) and concentrated. The crude product was co-evaporated with MeCN (3x) to remove traces of water. The resulting solid was repeatedly washed with EtOAc (20 mL) with stirring at 40 °C until no OSu ester was detected in the supernatant, giving a white solid.

[0287] Step 3: To a solution (0 °C) of the crude solid in MeOH / water (10 mL, 9:1) was added aqueous NaOH (2 M, 1.31 mL, 2.62 mmol), and the mixture was then stirred at room temperature for 45 min. The reaction was quenched with aqueous AcOH (1 M, 3.9 mL) at 0 °C. Methanol was then removed on a rotary evaporator and the aqueous suspension was diluted with water and filtered. The solid was washed with water and the aqueous phase was lyophilized to give intermediate XD56 (840 mg) as a white solid. In future reactions, quantitative conversion of crude XD56 was assumed in steps 1-3, corresponding to a purity of 35 wt %. MS (ESI + ) C 21 H 35 N3O6P + [M+H] + Calculated value: 456.2, actual value: 456.5.

[0288] Step 4: A portion of intermediate XD56 (490 mg crude, theoretical maximum 0.365 mmol) was suspended in DMF (4 mL) at room temperature. DIPEA (0.254 ml, 1.46 mmol) and 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid 2,5-dioxopyrrolidin-1-yl ester (146 mg, 0.424 mmol) in DMF (1 mL) were added and the mixture was stirred for 70 min. The mixture was concentrated and the crude product was immediately purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give the azide XD57 (163.6 mg, 64%) as a white solid after lyophilization. 1 H NMR (400 MHz, D2O) ppm = 7.52-7.41 (m, 4H), 5.40 (br t, J = 7.4 Hz, 1H), 4.94 (d, J = 7.8 Hz, 2H), 4.45 (q, J = 7.1 Hz, 1H), 4.17 (d, J = 7.1 Hz, 1H), 3.91 (s, 2H), 3.78 (t, J = 6.0 Hz, 2H), 3.71-3.61 (m, 10H), 3.51-3.44 (m, 2H), 2.69-2.55 (m, 2H), 2.28-2.16 (m, 2H), 2.16-2.04 (m, 1H), 1.77-1.65 (m, 2H), 1.58 (s, 3H), 1.49 (d, J = 7.3 Hz, 3H), 0.98 (br d, J = 6.6 Hz, 3H), 0.96 (br d, J = 6.4 Hz, 3H). MS (ESI - ) C 30 H 48 NO 10 P - [MH] - Calculated value: 683.3, actual value: 683.6.

[0289] B. Preparation of Linker-Drug Compound XD58 [ka]

[0290] Linker-Drug XD58 The azide XD57 (21.4 mg, 0.030 mmol) was reacted with alkyne XD43 according to general procedure XXE. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the linker-drug compound XD58 (19.7 mg, 67%) as a fluffy white solid after lyophilization. 1 H NMR (400 MHz, D2O) ppm = 7.92 (br s, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.70 (s, 2H), 5.30 (br t, J = 7.0 Hz, 1H), 4.83 (br d, J = 4.6 Hz, 2H), 4.49 (br t, J = 4.8 Hz, 2H), 4.35 (q, J = 7.1 Hz, 1H), 4.27 (br s, 2H), 4.08 (d, J = 7.0 Hz, 1H), 4.05 (br s, 2H), 3.87-3.82 (m, 2H), 3.81 (s, 2H), 3.67 (t, J = 5.9 Hz, 2H), 3.63-3.53 (m, 6H), 3.53-3.40 (m, 8H), 2.61-2.42 (m, 2H), 2.11 (br s, 2H), 2.06-1.92 (m, 1H), 1.69-1.54 (m, 2H), 1.49 (s, 3H), 1.38 (d, J = 7.1 Hz, 3H), 0.87 (d, J = 7.6 Hz, 3H), 0.85 (d, J = 7.4 Hz, 3H). MS (ESI - ) C 42 H 62 N8O 15 P - [MH] - Calculated value: 949.4, actual value: 949.8.

[0291] Example 19 Synthesis of Linker-Drug Compound XD59 A. Preparation of dialkyne linker XS30 [ka]

[0292] 1-(Prop-2-yn-1-yl)piperazine (XS28) Step 1: To a solution of piperazine-1-carboxylic acid tert-butyl ester (3.44 g, 18.5 mmol) in MeCN (17 mL) at 0° C., DIPEA (5.87 mL, 33.6 mmol) was added followed by propargyl bromide (80% in toluene, 1.80 mL, 16.8 mL). The reaction mixture was allowed to reach room temperature and stirred for 2 h. It was then partitioned between EtOAc (25 mL) and water (25 mL). The aqueous phase was extracted with EtOAc (12 mL) and the combined organic phase was washed with brine (30 mL), dried over Na2SO4 and concentrated. Flash chromatography (silica gel, 0-50% EtOAc in heptane) afforded 4-(prop-2-yn-1-yl)piperazine-1-carboxylic acid tert-butyl ester (3.56 g, 15.9 mmol, 94%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) ppm = 3.47 (t, J = 5.1 Hz, 4H), 3.32 (d, J = 2.4 Hz, 2H), 2.51 (t, J = 5.1 Hz, 4H), 2.26 (t, J = 2.4 Hz, 1H), 1.46 (s, 9H). MS (ESI + ) C 12 H 21 N2O2 + [M+H] + Calculated value 225.2, actual value 225.2.

[0293] Step 2 A portion of the product (2.82 g, 12.6 mmol) was dissolved in DCM (6.3 mL) and a solution of 4 M HCl in dioxane (28.3 mL, 113 mmol) was added dropwise with stirring. The reaction mixture was stirred at room temperature for 4 h. The resulting suspension was filtered and the residue was washed with DCM (2×5 mL). The white solid was dried in vacuum to give the amine XS28 (2.48 g, quantitative) as the hydrochloride salt. 1H NMR (400 MHz, D2O) ppm = 3.98 (d, J = 2.5 Hz, 2H), 3.52 (br s, 8H), 3.06 (t, J = 2.5 Hz, 1H). MS (ESI + ) C7H 13 N2 + [M+H] + Calculated value: 125.1, actual value: 125.1.

[0294] ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid allyl ester (XS29) To a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (1.90 g, 9.00 mmol) in DCM (45 mL) was added DIPEA (6.28 mL, 36.0 mmol) followed by HATU (3.59 g, 9.45 mmol). The reaction mixture was stirred for 1 h and then amine XS28 (1.87 g, 9.45 mmol) was added. The reaction mixture was stirred for 30 min and then partitioned between EtOAc (50 mL) and saturated aqueous NaHCO3 (50 mL). The aqueous phase was extracted with EtOAc (2 x 50 mL) and the combined organic phase was washed with water (50 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography (silica gel, 0-0.1% Et3N in EtOAc) to give the amide XS29 (2.20 g, 77%) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.00 (s, 2H), 3.43 (d, 4H), 3.38 (t, J = 7.1 Hz, 2H), 3.29 (d, J = 2.4 Hz, 2H), 3.16 (t, J = 2.4 Hz, 1H), 2.45-2.33 (m, 4H), 2.26 (t, J = 7.5 Hz, 2H), 1.54-1.42 (m, 4H), 1.29-1.25 (m, 2H). MS (ESI + ) C 17 H 24N3O3 + [M+H] + Calculated value: 318.2, actual value: 318.3.

[0295] 4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-1,1-di(prop-2-yn-1-yl)piperazin-1-ium bromide (XS30) To a solution of amine XS29 (0.528 g, 1.66 mmol) in MeCN (3.3 mL) at 0 °C was added propargyl bromide (80%, 8.32 mmol in toluene 0.926 mL). The reaction mixture was allowed to reach room temperature and stirred overnight, then added dropwise to ether (45 mL) with stirring. An oily precipitate formed and the supernatant was decanted off. The residue was washed with ether (5 mL) and subsequently dissolved in a MeCN / toluene (4:1) mixture and concentrated to give the quaternary amine XS30 (0.710 g, 79%) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.01 (s, 2H), 4.59 (d, J = 2.1 Hz, 4H), 4.15 (t, J = 2.2 Hz, 2H), 3.90-3.79 (m, 4H), 3.60-3.48 (m, 4H), 3.39 (t, J = 7.1 Hz, 2H), 2.34 (t, J = 7.4 Hz, 2H), 1.55-1.44 (m, 4H), 1.30-1.25 (m, 2H). MS (ESI + ) C 20 H 26 N3O3 + [M] + Calculated value: 356.2, actual value: 356.4.

[0296] B. Preparation of Linker-Drug Compound XD59 [ka]

[0297] Linker-Drug XD59 Water was purged with nitrogen gas for 20 min with stirring before use. Alkyne XS30 (9.2 mg, 0.017 mmol) in THF / water (1:10, 0.55 mL) was added to solid XD57 (30.1 mg, 0.043 mmol) at room temperature under nitrogen atmosphere. Copper(II) sulfate pentahydrate (8.3 mg, 0.033 mmol) in water (1.0 mL) was then added to give a clear solution. The headspace of the vial was purged with nitrogen gas, followed by the addition of sodium ascorbate (0.013 g, 0.064 mmol) in water (0.48 mL) at room temperature to give an opaque suspension. Further alkyne XS30 (10 mg in THF / water (1:10, 0.540 mL)) was added in four portions over 3 h, at which point UPLC-MS analysis showed complete conversion. THF was removed by brief rotary evaporation and the aqueous solution was diluted with 10% MeCN in 25 mM NH4HCO3 (10 mL) and purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in milliQ water, 90:10 to 50:50 gradient) to give the linker-drug compound XD59 (23.0 mg) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 8.56 (s, 2H), 7.46 (d, J = 8.5 Hz, 4H), 7.41 (d, J = 8.5 Hz, 4H), 6.81 (s, 2H), 5.39 (br t, J = 6.9 Hz, 2H), 4.89 (br d, J = 7.0 Hz, 4H), 4.74 (s, 4H), 4.69 (br t, J = 4.8 Hz, 4H), 4.42 (q, J = 7.1 Hz, 2H), 4.16 (d, J = 6.9 Hz, 2H), 4.10-4.01 (m, 4H), 3.98 (br t, J = 4.7 Hz, 4H), 3.90 (s, 4H), 3.74 (t, J = 5.9 Hz, 4H), 3.68-3.62 (m, 4H), 3.62-3.52 (m, 14H), 3.52-3.43 (m, 4H), 2.68-2.52 (m, 4H), 2.42 (br t, J = 7.4 Hz, 2H), 2.26-2.14 (m, 4H), 2.09 (dq, J = 13.6, 6.8 Hz, 2H), 1.67 (dt, J = 16.3, 8.2 Hz, 4H), 1.60-1.51 (m, 10H), 1.46 (d, J = 7.3 Hz, 6H), 1.34-1.20 (m, 2H), 0.96 (d, J = 7.1 Hz, 6H), 0.93 (d, J = 7.1 Hz, 6H). MS (ESI - ) C 80 H 122 N 15 O 23 P2 - [MH] - The calculated value is 1722.8 and the measured value is 1723.2.

[0298] Example 20 Synthesis of リンカー-drug compound XD63 Manufactured by A ホスホン acid エステルXD61

change

[0299] (E)-(5-((tert-butyldiphenylsilyl)oxy)-4-methylpent-3-en-1-yl)phosphonic acid dimethyl ester (XL1) Methylphosphonic acid dimethyl ester (13.1 ml, 121 mmol) was dissolved in THF (440 mL), cooled to -78°C, and n-butyl lithium (75 ml, 121 mmol) was added. The mixture was stirred at -78°C for 1 h, then warmed to -50°C, followed by the addition of CuI (11.5 g, 60.4 mmol) and stirring at this temperature for 1 h. The mixture was cooled again to -78°C, and XD51 (19.7 g, 54.9 mmol) dissolved in THF (110 mL) was added. The reaction was allowed to warm to room temperature overnight, then quenched with saturated aqueous NH4Cl (500 mL). The mixture was extracted with EtOAc (2 x 500 mL) and the combined organic phase was washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 0 to 100% EtOAc in heptane) to afford XL1 (23.2 g, 95% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.69-7.63 (m, J = 7.9, 1.5 Hz, 4H), 7.44-7.34 (m, 6H), 5.46-5.41 (m, 1H), 4.04 (br s, 2H), 3.75 (s, 3H), 3.73 (s, 3H), 2.38-2.28 (m, 2H), 1.83-1.73 (m, 2H), 1.60 (s, 3H), 1.06 (s, 9H). MS (ESI+)C 24 H 36 O4PSi + [M+H] + Calculated value: 447.2, actual value: 447.4.

[0300] B. Preparation of Linker-Drug Compound XD63 (E)-(5-((tert-butyldiphenylsilyl)oxy)-4-methylpent-3-en-1-yl)phosphonic acid bis(2-cyanoethyl)ester (XD60) Phosphonate ester XL1 (580 mg, 1.30 mmol) was converted to the phosphonic acid dichloride as described in general procedure XXB. The crude product was then reacted with 3-hydroxypropionitrile as described in the synthesis of XD21, except that 2,6-lutidine was used instead of pyridine. The crude product was purified by flash chromatography (silica gel, 20-100% EtOAc in heptane) to give phosphonate ester XD60 (360 mg, 53%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.69-7.62 (m, 4H), 7.46-7.34 (m, 6H), 5.44 (br t, J = 7.0 Hz, 1H), 4.34-4.20 (m, 4H), 4.05 (s, 2H), 2.74 (t, J = 6.1 Hz, 4H), 2.45-2.33 (m, 2H), 1.97-1.83 (m, 2H), 1.61 (s, 3H), 1.06 (s, 9H). MS (ESI + ) C 28 H 37 N2NaO4PSi + [M+H] + Calculated value: 547.2, actual value: 547.5.

[0301] (E)-(5-((tert-butyldiphenylsilyl)oxy)-4-methylpent-3-en-1-yl)phosphonic acid 2-cyanoethyl ester triethylamine (XD61) DBU (0.053 ml, 0.349 mmol) was added to a solution of phosphonate ester XD60 (122 mg, 0.233 mmol) in THF (2 mL) at room temperature. After stirring for 30 min, the mixture was concentrated to about 0.5 mL and the solution was diluted with MeOH (1 mL). The solution was then diluted with a short DOWEX 50WX8 (H + The DBU was removed by passing it through a plug of hexane (type 2) and eluting the product with methanol.

[0302] Triethylamine (0.032 ml, 0.233 mmol) was added to the eluate and the mixture was concentrated and coevaporated with MeCN (2x). The phosphonate XD61 (120 mg, 97%) was isolated as the triethylamine salt in a phosphonate:EtN ratio of 1:0.6. 1 H NMR (400 MHz, CD3OD) ppm = 7.71-7.62 (m, 4H), 7.46-7.36 (m, 6H), 5.43 (td, J = 7.3, 1.3 Hz, 1H), 4.08 (q, J = 6.7 Hz, 2H), 4.05 (s, 2H), 3.20 (q, J = 7.3 Hz, 4H), 2.78 (t, J = 6.1 Hz, 2H), 2.38-2.28 (m, 2H), 1.65 (s, 3H), 1.71-1.61 (m, 2H), 1.31 (t, J = 7.3 Hz, 5H), 1.04 (s, 9H). MS (ESI - ) C 25 H 33 NO4PSi - [MH] - Calculated value: 470.2, actual value: 470.4.

[0303] [ka]

[0304] ((2S)-1-(((2S)-1-((4-((((2-cyanoethoxy)((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (XD62) Step 1 Phosphonate ester XD61 (187 mg, 0.326 mmol) and Fmoc-Val-Cit-PAB (295 mg, 0.490 mmol) were combined in a round bottom flask and coevaporated with DMF (3× 8 mL). DMF (3.2 mL) was then added under nitrogen atmosphere followed by PyBOP (255 mg, 0.490 mmol) and DIPEA (0.057 ml, 0.326 mmol) at room temperature. After 5 min, DIPEA (0.114 ml, 0.653 mmol) was further added and the mixture was stirred for 2 h. The reaction mixture was then slowly added dropwise to water (70 mL, 0° C.). Stirring should be gentle to avoid gel formation. The white suspension was gently stirred for 5 min and then filtered. The solid was collected and coevaporated with MeCN (2×) to remove traces of water. The solid was purified by flash chromatography (silica gel, 0-12% MeOH in DCM) to give the product (263 mg, 76%) as a white solid. MS (ESI + ) C 58 H 72 N6O9PSi + [M+H] + Calculated value: 1055.5, actual value: 1056.0.

[0305] Step 2 A portion of the product (257 mg, 0.244 mmol) was suspended in THF (3.8 mL) and pyridine (0.370 mL) in a PFA vial under nitrogen. The vial was cooled to 0 °C and HF·pyridine (0.25 ml, 70%) was added. The mixture was stirred at this temperature for 6 h, then the cooled suspension was carefully added to a mixture of saturated aqueous NaHCO3 / 10% iPrOH in EtOAc (0 °C). After stirring for 5 min, the phases were separated and the organic phase was washed with HCl (1 M) and brine, dried over Na2SO4, filtered and concentrated onto silica gel. Flash chromatography (silica gel, 0–15% MeOH in DCM) afforded the alcohol XD62 (148 mg, 74%) as a white solid. 11H NMR (400 MHz, DMSO-d6) ppm = 10.10 (s, 1H), 8.12 (br d, J = 7.5 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.45 - 7.37 (m, 3H), 7.36 - 7.29 (m, 4H), 5.97 (br t, J = 5.7 Hz, 1H), 5.40 (s, 2H), 5.34 (td, J = 7.1, 1.0 Hz, 1H), 5.03 - 4.91 (m, 2H), 4.67 (t, J = 5.6 Hz, 1H), 4.46 - 4.37 (m, 1H), 4.34 - 4.18 (m, 3H), 4.14 - 4.04 (m, 2H), 3.98 - 3.87 (m, 1H), 3.75 (d, J = 5.5 Hz, 2H), 3.07 - 2.90 (m, 2H), 2.88 (t, J = 5.9 Hz, 2H), 2.27 - 2.12 (m, 2H), 2.05 - 1.93 (m, 1H), 1.91 - 1.77 (m, 2H), 1.75 - 1.54 (m, 2H), 1.51 (s, 3H), 1.49 - 1.29 (m, 2H), 0.88 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 42 H 54 N6O9P + [M+H] + Calculated value: 817.4, Measured value: 817.8.

[0306] Linker - Drug XD63 Deprotection of phosphonate ester XD62 with NaOH, followed by amide coupling with 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester and purification by RP-HPLC was carried out as in steps 3 and 4 of the synthesis of XD57 with the following modifications: in step 3, 5 equivalents of NaOH (2M) were used and the reaction time was 2 h. In step 4, 2 equivalents of OSu ester were used. Linker-drug XD63 (47.9 mg, 36%) was obtained as a white solid. 1 H NMR (400 MHz, D2O) ppm = 7.46 (s, 4H), 6.82 (s, 2H), 5.40 (br t, J = 7.1 Hz, 1H), 4.90 (br d, J = 7.5 Hz, 2H), 4.45 (br t, J = 6.9 Hz, 1H), 4.10 (br d, J = 7.9 Hz, 1H), 3.91 (s, 2H), 3.46 (br t, J = 6.9 Hz, 2H), 3.14 (br t, J = 6.8 Hz, 2H), 2.31 (br t, J = 6.6 Hz, 2H), 2.24-2.13 (m, 2H), 2.11-2.00 (m, 1H), 1.99-1.75 (m, 2H), 1.71-1.47 (m, 11H), 1.31-1.19 (m, 2H), 0.95 (br dd, J = 6.4, 2.8 Hz, 6H). MS (ESI + ) C 34 H 52 NO 10 P + [M+H] + Calculated value: 735.4, actual value: 735.7.

[0307] Example 21 Preparation of Linker-Drug Compound XD65 [ka]

[0308] ((2S)-1-(((2S)-1-((4-((((((E)-4-((tert-butyldiphenylsilyl)oxy)-3-methylbut-2-en-1-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (XD64) Fmoc-Val-Cit-PAB (300 mg, 0.499 mmol) was dissolved in DMF (7.0 mL) at room temperature under nitrogen atmosphere, and (3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (0.174 ml, 0.548 mmol) was added, followed by dropwise addition of tetrazole in MeCN (0.45 M, 1.22 ml, 0.548 mmol). The mixture was stirred at room temperature for 2 h. Meanwhile, alcohol XD47 (282 mg, 0.828 mmol, Serra, S. Tetrahedron: Asymmetry, 2014, 25, 1561-1572) and 5-(ethylthio)-1H-tetrazole (143 mg, 1.097 mmol) were placed in a 10 mL round bottom flask and coevaporated with dry MeCN. The residue was dissolved in DMF (0.5 mL) under nitrogen atmosphere and the mixture was then added using a cannula at room temperature. After stirring overnight, tBuOOH (5.5 M in decane, 0.199 ml, 1.097 mmol) was added at 0 °C and after 2 min the reaction was warmed to room temperature and stirred for 90 min. The reaction was then poured into ice-cold water (70 mL) with stirring and after 5 min the suspension was filtered and washed with a small amount of water (2 × 6 mL). The solid was then purified by flash chromatography (silica gel, 0-10% MeOH in DCM) to give a mixture of product and Fmoc-Val-Cit-PAB (354 mg). This material was used in the next step without further purification. MS (ESI + ) C 57 H 69 N6NaO 10 PSi + [M+Na] + Calculated value: 1079.5, actual value: 1079.9.

[0309] Linker-Drug XD65 ((2S)-1-(((2S)-1-((4-((((((E)-4-((tert-butyldiphenylsilyl)oxy)-3-methylbut-2-en-1-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (162 mg, 0.153 mmol) was dissolved in DMF (3.0 mL). TBAF (1.0 M in THF, 458 μl, 0.458 mmol) was added at room temperature. After 35 min, more TBAF (1.0 M in THF, 1.30 mL, 1.30 mmol) was added and the reaction was continued for 45 min. Ether (approximately 50 mL) was added to give a cloudy oil. The supernatant was removed and the remaining oil was treated twice with ether (add ether, stir, decant). The oily residue was dissolved in DMF (0.3 mL) and acetic acid (0.044 mL, 0.764 mmol) was added, followed by DIPEA (0.080 mL, 0.458 mmol) and 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (70.6 mg, 0.229 mmol) at room temperature. After 25 min, the reaction was observed to be completely complete. Acetic acid (0.044 mL, 0.764 mmol) was added and the mixture was concentrated. The crude product was purified by preparative RP-HPLC (Milli-Q water × 0.1% TFA / MeCN, 90:10 to 40:60 gradient) and the product fraction was lyophilized. Partial degradation was observed upon lyophilization under such acidic conditions. A portion of the impure product was purified again by preparative RP-HPLC (10 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 65:35 gradient) to give the linker-drug compound XD65 (15.3 mg) after lyophilization. 1H NMR (400 MHz, DMSO-d6) ppm = 10.09 (s, 1H), 8.12 (br d, J = 7.6 Hz, 1H), 7.85 (br d, J = 8.8 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 7.00 (s, 2H), 6.11-6.03 (m, 1H), 5.58-5.50 (m, 1H), 5.45 (br s, 2H), 4.75 (br d, J = 6.8 Hz, 2H), 4.43-4.28 (m, 3H), 4.19 (dd, J = 8.7, 6.9 Hz, 1H), 3.78 (s, 2H), 3.39-3.34 (m, 2H), 2.97 (dtt, J = 18.9, 12.8, 6.3 Hz, 2H), 2.23-2.06 (m, 2H), 2.02-1.91 (m, 1H), 1.78-1.65 (m, 1H), 1.65-1.29 (m, 10H), 1.22-1.12 (m, 2H), 0.85 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H). MS (ESI - ) C 33 H 48 NO 11 P - [MH] - Calculated value: 735.3, actual value: 735.9.

[0310] Example 22 Synthesis of Conjugates from Linker-Drug Compounds (LD) The ADC numbers in Table 1 indicate the antibodies used with the corresponding linker-prodrugs synthesized as disclosed in the Examples.

[0311] The antibodies used in the conjugates shown in Table 1 are as follows: · Anti-CD20 monoclonal antibody (MoAb) Rituximab (r) Anti-Her2 MoAb trastuzumab (t) or trastuzumab-41C (t) in which the amino acid at position 41 of the antibody heavy chain (HC) is replaced by cysteine 41C ) Anti-CD123 MoAb (Byondis' proprietary MoAb CD123) anti-5T4 MoAb (5T4), which is the anti-5T4 antibody disclosed in WO 2015 / 177360 as H8-HC41C (the heavy chain comprises the amino acid sequence shown in SEQ ID NO:8 and the light chain comprises the amino acid sequence shown in SEQ ID NO:11); anti-PSMA MoAb SYD1030 41C(p), an anti-PSMA antibody with an engineered cysteine ​​at position 41 (i.e., HC41C) of the heavy chain disclosed in WO 2015 / 177360 as SYD1030 (heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and light chain comprising the amino acid sequence set forth in SEQ ID NO:5); 41C ) · Isotype control antibody (i).

[0312] The isotype control antibodies used had the variable domain sequence of the human anti-HIV1 pg120 antibody B12, accession number 2NY7 (Zhou et al., 2007, Nature, 445, 732-737), which was used as an IgG1κ isotype control antibody (accession numbers P01857 and P01834, respectively).

[0313] Each conjugate was synthesized according to the method described in Examples 22a-c. All conjugates with DAR less than 8 shown in Table 1 were synthesized according to the procedure described in Example 22a, except for the conjugate with DAR2, which was prepared by site-specific conjugation as described in Example 22b. Conjugates with high DAR (8 or higher) were prepared according to the procedure described in Example 22c. The DAR (pAg-antibody ratio in the conjugate) is also shown in Table 1.

[0314] Example 22a Synthesis of DAR2 conjugates To a solution of antibody (10-12 mg / mL), TRIS (1% by volume, 1 M, pH 8), EDTA (4% by volume, 25 mM) and TCEP (5 mM in water) were added. The resulting solution was incubated for 2 hours at room temperature. After incubation, the reduced antibody was rebuffered with 4.2 mM histidine, 50 mM trehalose (pH 6) and treated with dimethylacetamide (DMA) and linker-drug compound (LD) (10 mM in DMA, ≥ 1.5 equivalents per SH). The final DMA content was approximately 10% by volume. The resulting mixture was roller mixed overnight at room temperature in the dark. Activated charcoal was added and the suspension roller mixed in the dark for 1 hour, filtered and washed with 4.2 mM histidine, 50 mM trehalose (pH 6). The solution was rebuffered with 4.2 mM histidine, 50 mM trehalose, pH 6, and sterile filtered.

[0315] Example 22b Synthesis of conjugates of DAR2 by site-specific conjugation A part of the DAR2 complex (ADC-XD18-CD123 shown in Table 1) 41C , ADC-XD18-5T4 41 ) were synthesized by site-specific conjugation, with the linker-drug molecule being attached only through an engineered cysteine ​​at position 41 ("41C") of the antibody heavy chain according to the Kabat numbering system. These conjugates were prepared by the methods disclosed in WO 2015 / 177360 and WO 2017 / 137628.

[0316] Example 22c Synthesis of complexes with high DAR (DAR8, DAR16, and DAR10, DAR20) To a solution of antibody (12 mg / mL in 4.2 mM histidine, 50 mM trehalose, pH 6) was added EDTA (25 mM in water, 4% v / v) and TRIS (1 M in water, pH 8, 2% v / v).

[0317] For conjugates DAR8 or DAR16, a wild-type antibody was used. For conjugates DAR10 or DAR20, a 41C-modified antibody was used in which the amino acid at position 41 in the Kabat numbering system of the heavy chain was replaced with a cysteine. This modification introduces two additional cysteines into the amino acid sequence of the antibody, which are reduced with TCEP in the next step, resulting in a total of 10 positions available for conjugation with the linker-drug (LD).

[0318] TCEP (10 mM in water, 30 equivalents) was added and the mixture was incubated overnight at room temperature. The reaction was cleared with a centrifugal concentrator (Vivaspin filter, molecular weight cutoff 30 kDa, PES) using 4.2 mM histidine, 50 mM trehalose, pH 6.

[0319] DMA was added, followed by the linker-drug solution. For DAR8 and DAR16 conjugates, 10 mM, 16 equivalents were added in DMA. For DAR10 and DAR20 conjugates, 10 mM, 20 equivalents were added in DMA.

[0320] Conjugates of DAR16 and DAR20 were prepared using a linker-drug with branched linkers, each with two pAgs (Linker-drug XD59 shown in Table 1). The final concentration of DMA was 10%.

[0321] The resulting mixture was incubated at room temperature for 3 hours or overnight, protected from light. To remove excess linker-drug, activated charcoal was added and the mixture was incubated at room temperature for 1 hour. Activated charcoal was removed using a 0.2 mm PES or PVDF filter, and the resulting ADC was added to 4.2 mM histidine, 50 mM trehalose (pH 6) using a Vivaspin centrifugal concentrator (molecular weight cutoff 30 kDa, PES). Finally, the ADC solution was sterile filtered using a 0.2 mm PVDF filter.

[0322] To approximate the DAR (pAg antibody ratio) of the conjugate synthesized as described in Example 22a or 22b to the target DAR of 2, alternative conjugation was performed using hydrophobic seco-DUBA (SYD980, described in WO 2015 / 177360), which enables simple determination of DAR by HIC.

[0323] The approximate DAR obtained for the conjugate with a target DAR of 2 is shown in Table 1. For the high-DAR conjugate synthesized as described in Example 22c, the target DAR is shown as the "target value" in Table 1. The actual DAR may deviate somewhat from this value (this means that the peaks overlap (for the ADC with a target DAR of 2) or the manufactured ADC is not fully reduced / conjugated (for the ADCs with target DARs of 8, 10, 16, and 20), so it could not be measured by standard methods (HIC)).

[0324] In the case where multiple values are listed separated by commas for DAR or %HMW, these refer to different batches of the same ADC.

[0325] In Table 1, "<LOD" means below the limit of detection.

[0326]

Table 1-1

[0327]

Table 1-2

[0328]

Table 1-3

[0329]

Table 1-4

[0330] [Table 1-5]

[0331] [Table 1-6]

[0332] Example 23: Activity of prodrugs and conjugates against γδ T cells To determine the activity of (unconjugated) phosphoantigen prodrugs, Raji target cells were preincubated with XC1 and XD1 before co-culture with peripheral blood mononuclear cells (PBMCs) containing effector cells. Selective γδ T cell activation was examined in vitro after co-culture of PBMCs with tumor cells (derived from the CD20-positive Burkitt's lymphoma human tumor cell line Raji) pretreated with the phosphoantigen HMBPP, the phosphoantigen prodrugs XC1, XD1, or the conjugates listed in Table 1. The structures of the tested prodrugs are shown in Table 2.

[0333] [Table 2]

[0334] PBMCs from healthy human donors were used as a source of immune cells.

[0335] Once activated, Vγ9Vδ2 γδ T cells produce cytokines to activate immunity and release cytotoxic granules (degranulation) to kill target cells. Only Vγ9Vδ2 γδ T cells are known to sense fluctuations in phosphoantigen levels, but some of the induced effector mechanisms are shared with other immune cells, including CD8+ T cells, NK cells and other subsets of γδ T cells. All these immune cell populations are present in PBMCs isolated from the blood of healthy donors. Therefore, PBMCs are a good source of cells to perform in vitro experiments and determine the selective activation of γδ T cells.

[0336] Identification of different immune cell populations can be achieved by specific staining with fluorescently labeled monoclonal antibodies. When monensin and / or brefeldin A are added to the co-culture of PBMCs and targets, the produced IFNγ is captured by the activated cells. Staining with fluorescently labeled antibodies in the presence of saponin, which allows the anti-IFNγ antibodies to be internalized, identifies IFNγ producing cells. Fluorescently labeled antibodies against CD107a can also be added to the co-culture to stain cells undergoing degranulation. Thus, in combination with fluorescently labeled immune cell specific markers, CD107a and IFNγ markers, it is possible to determine the activation state of γδ T cells and / or other immune cell subsets after co-culture with pretreated target cells.

[0337] Materials and Methods The CD20-positive Burkitt's lymphoma human tumor cell line Raji (DSMZ, the German collection of Microorganisms and cell cultures GmbH (Leibniz Institute, Germany)) was used for in vitro experiments. Raji cells were cultured in complete growth medium (CGM) (RPMI1640 (Lonza, Walkersville, MD, USA) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco- Life Technologies; Carlsbad, CA) and 80 U / mL penicillin-streptomycin solution (Lonza Group Ltd, Basel Switzerland)). Raji cells were maintained at 37°C in a humidified incubator with 5% CO2 and passaged twice a week.

[0338] For stimulation with the conjugates (ADCs) of the invention, unconjugated phosphoantigen prodrugs and HMBPP, Raji cells were harvested and 5×10 6The cells were diluted to 1000 cells / mL and 50 μL of this cell suspension (corresponding to 250,000 cells / well) was seeded into a 96-well plate. Serial 10-fold dilutions of 2-fold concentrated prodrugs and ADCs were prepared in CGM. The seeded Raji cells were incubated with 50 μL / well of serially diluted compounds overnight at 37° C. in a humidified incubator with 5% CO2.

[0339] The following day, to remove excess unbound compound, the 96-well plates containing Raji cells and compounds (ADC, unbound phosphoantigen prodrug or HMBPP) were washed with 100 μL of CGM per well, centrifuged at 300×g for 3 minutes at room temperature, and the supernatant was removed.

[0340] As a source of immune cells, frozen peripheral blood mononuclear cells (PBMCs) from healthy human donors were thawed, resuspended in CGM, and placed in a humidified incubator at 37°C and 5% CO2 overnight to allow cells to recover.

[0341] Collect recovered PBMCs, count and culture at 10 x 10 in CGM. 6 Dilute to 50 µL / well (0.5 x 10 cells / mL). 6 100 μL / well equivalent to 1000 cells / well were added to Raji cells. A 2-fold concentrated anti-CD107a-AlexaFluor 647 solution was prepared in CGM containing GolgiStop (monensin) and GolgiPlug (brefeldin A) (BD Biosciences, San Jose, CA, USA) and 50 μL / well was added to Raji-PBMC co-cultures. In all experiments, 1% phytohemagglutinin (PHA-M, Gibco-ThermoFisher), a known non-specific activator of immune cells, was also included in the wells as a positive control. Samples were incubated for 6 h in a humidified incubator at 37 °C and 5% CO2.

[0342] For specific staining of immune cell subsets, a multicolor antibody staining cocktail was prepared in Brilliant Stain Buffer containing anti-CD3 BUV396 (not all inclusive), anti-CD8 BV421, anti-CD56 PE-Cy7, Viability Stain 780 (BD Biosciences, San Jose, CA, USA), anti-TCR V51 PerCP-Vio700, FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), and anti-TCR V52 BV711 (Biolegend, San Diego, USA). After 6 h of incubation, the plates were centrifuged at 300×g for 3 min at room temperature and the supernatant was discarded. The pellet was resuspended in 50 μL of antibody cocktail and incubated on ice for 30 min in the dark. Plates were washed twice with 100 μL ice-cold FACS buffer (PBS 1×, 0.1% v / w BSA, 0.02% v / v sodium azide (NaN3)), followed by centrifugation at 300×g for 3 min and discarding the supernatant. Cells were fixed and permeabilized with 100 μL / well Cytofix / Cytoperm solution (BD bioscience, San Jose, CA, USA) and incubated on ice for 20 min protected from light. Cells were washed three times with 150 μL BD Perm / wash solution containing saponin (10× BD Perm / Wash buffer was diluted with distilled water to make a 1× solution before use) followed by centrifugation at 300×g for 3 min and discarding the supernatant. Finally, cells were resuspended in FACS buffer and stored overnight in a refrigerator at 4° C. protected from light.

[0343] On day 3, stained PBMC / Raji cells were washed once with 150 μL of BD Perm / wash solution, followed by centrifugation at 300×g for 3 min, and the supernatant was discarded. The pellet was resuspended in 50 μL of a mixture of anti-IFNγ BV650 (BD Biosciences, San Jose, CA, USA) diluted in Perm / Wash solution and incubated on ice for 30 min in the dark. After incubation, the plate was washed once with 150 μL of ice-cold FACS buffer, followed by centrifugation at 300×g for 3 min, and the supernatant was discarded. The cell pellet was then resuspended in 150 μL of FACS buffer, and samples were analyzed on a BD FACSymphony A3 cell analyzer (BD Biosciences, San Jose, CA, USA) equipped with a high-throughput sampler to analyze 96-well plates.

[0344] Gating Strategy Analyzed with FlowJo V10.7, the resulting samples were gated to identify different immune cell populations (Figure 1). First, a time gate was applied to ensure constant flow (Figure 1A) and eliminate potential irregularities, and then doublets and dead cells were excluded in FSC-A vs. FSC-H and SSC-A vs. SSC-H plots (Figure 1B and Figure 1C).

[0345] Viable cells were selected (Figure 1D), followed by lymphocyte selection based on FSC / SSC (Figure 1E). CD3-negative CD56-positive cells were then identified as NK cells (Figure 1F). CD3-positive cells were further divided into Vδ2-positive and Vδ1-positive cells (Figure 1G). Lymphocytes were also subdivided into CD8-positive cytotoxic T cells (Figure 1H), resulting in four families: - NK cells are CD3 - CD56 + Identified as cells, - CD8 + T cells are CD3 + Vδ1 - Vδ2 - CD8 + Identified as, - Vδ1 γδ T cells are CD3 +Vδ1 + Vδ2 - Identified as, - Vδ2 γδ T cells are CD3 + Vδ1 - Vδ2 + Identified as.

[0346] Of note, the B6 clone used here to stain Vδ2 γδ T cells only stains Vγ9Vδ2 γδ T cells, not the Vγ9 subunit of Vδ2 γδ T cells. - For these four immune cell populations, CD107a + or IFNγ + The percentage of CD107 cells was determined. + or IFNγ + The median fluorescence intensity of the cell population was determined as a measure of activity per cell. CD107a has been described as a marker for degranulation and strongly correlates with target cell killing. IFNγ accumulation caused by Brefeldin A / Monensin treatment, which prevents secretion, is a measure of IFNγ cytokine production.

[0347] Results / Conclusion Cell membrane-permeable prodrugs induce activation of Vδ2 γδ T cells Neither the prodrugs XC1 nor XD1 inhibited other immune cells (NK cells, CD8 + XC1 and XD1 pretreated Raji cells dose-dependently induced IFNγ production and degranulation (i.e., CD107a) of Vδ2 γδ T cell populations in primary human PBMCs (Fig. 2A, C), without inducing direct activation of Vδ2 γδ T cells (Vδ1 γδ T cells) (Fig. 2B, D). The positive control phytohemagglutinin (PHA-M) was able to activate all immune cells (Fig. 3A-3D, 3I-3L), indicating that they retain their immunogenic potential. IFNγ production and degranulation of Vδ2 γδ T cells were more potently induced by XC1 and XD1 pretreated Raji cells compared to pretreatment with HMBPP (Fig. 2A, C).

[0348] The three prodrugs were conjugated to rituximab or a non-binding isotype control via a cleavable linker as described in Example 4. Raji cells pre-incubated with the CD20-targeting ADC-XC4-r, ADC-XD4-r and ADC-XD13-r suppressed IFNγ production by V52γδ T cells with mean EC 50 and degranulation (CD107a) with mean EC51.3ng / ml, 45.5, and 470ng / ml, respectively. 50 and ADC-XC4-i and ADC-XD4-i were less effective at inducing V52 γδ T cell activation, and the non-binding control ADC-XD13-i did not induce IFNγ production or degranulation in V52 γδ T cells, so potent V52 γδ T cell activation was dependent on target cell-mediated ADC activation (Figure 4).

[0349] As expected, Raji cells pretreated with the CD20-binding antibody rituximab also activated V52γδ T cells, however rituximab-ADC induced degranulation and IFNγ production in more V52γδ T cells than rituximab itself (Fig. 5C,D), as well as higher CD107a and IFNγ expression per cell (Fig. 5E,F,6).

[0350] Pretreatment of Raji cells with rituximab also activated NK cells, likely via FcγR, which is well known to be expressed on NK cells. Pretreatment of Raji cells with ADC did not further enhance the percentage of activated NK cells (Figure 4B, Figure 4F). These results indicate that pretreatment of tumor cells with CD20-binding ADC resulted in IFNγ induction and degranulation of Vδ2γδ T cells in a dose-dependent and more potent manner than rituximab, and that the ADC of this application can enhance anti-tumor immune responses depending on target binding and internalization. The ADC has an active Fc tail that likely activates other immune cells through well-described FcγR interactions.

[0351] [Table 3]

[0352] Example 24 Vδ2 γδ T cell activity induced by various pAg complexes Several synthetic phosphoantigen (pAg) conjugates were conjugated with rituximab (anti-CD20) and tested for their ability to selectively activate V52 γδ T cells after overnight incubation with CD20 positive Raji cells. The conjugates tested are listed in Table 1. Pretreated Raji cells were co-cultured with PBMCs and activation (IFNγ and TNFα production) and degranulation (CD107a) of V52 γδ T cells, V51 γδ T cells, CD8 positive T cells and NK cells were determined using multicolor flow cytometry as described in Example 23.

[0353] Materials and Methods cell binding Raji cells were cultured as described in Example 23. For cell binding in 96-well plates, 100,000 Raji cells / well were washed twice with ice-cold FACS buffer (PBS 1x, 0.1% v / w BSA, 0.02% v / v sodium azide (NaN3)) followed by addition of 50 μL per well of various concentrations of pAg conjugates, antibodies (e.g. rituximab) alone, or non-binding isotype control pAg conjugates diluted in ice-cold FACS buffer. After 30 min incubation at 4°C, cells were washed twice with ice-cold FACS buffer. Then, 50 μL per well of APC-conjugated secondary F(ab')2 goat anti-human IgG (Fc fragment specific, Jackson Immuno research, 109-136-098, 1:6000 or 1:500) was added. After 30 min at 4°C, cells were washed twice and resuspended in 150 μL of ice-cold FACS buffer. Fluorescence intensity was determined by flow cytometry using a FACSVerse or FACSymphony (BD Biosciences) and expressed as median fluorescence intensity (MFI). Curves were fitted by nonlinear regression with variable slope (4 parameters) in GraphPad Prism version 9. EC50 was calculated in GraphPad Prism as the concentration (μg / mL) midway between the response at the bottom and top of the curve. Binding experiments were performed in duplicate or triplicate.

[0354] Determination of direct compound-associated cell death Raji cells were seeded in CGM (complete growth medium, RPMI-1640 (Gibco-Life Technologies; Carlsbad, CA), supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco-Life Technologies; Carlsbad, CA) and 80 U / mL penicillin-streptomycin solution (Gibco-Life Technologies; Carlsbad, CA)) in 96-well plates (90 μL / well, 1000 cells / well) or 384-well plates (45 μL / well, 500 cells / well) and incubated at 37°C in a humidified incubator with 5% CO2. After overnight incubation, 10 μL or 5 μL of various concentrations of antibodies (e.g., rituximab), pAg conjugates or control compounds (toxin (duocarmycin type): cyclopropyl DC1) were added. After 6 days, metabolic activity was assessed using the CellTiter-Glo™ (CTG) Luminescence Assay Kit from Promega Corporation (Madison, WI) according to the manufacturer's instructions. Cell viability was expressed as the percentage of viability relative to the mean of untreated or vehicle-treated cells (growth medium only or 1% DMSO) multiplied by 100. Potency was calculated by subtracting the bottom of the dose-response curve (DRC) from 100%.

[0355] Functional assays (determination of γδ T cell activity induced by different pAg complexes) Functional assays were performed as disclosed in Example 23. In most experiments, in addition to anti-IFNγ BV650, anti-TNFα PE (BD Biosciences, San Jose, CA, USA, clone Mab11) was also included in the intracellular cytokine staining step. The highest compound concentration used for pretreatment of Raji cells was 150 μg / mL for antibody / conjugate. EC50 was calculated in GraphPad Prism as the concentration (μg / mL) midway between the bottom and top of the curve response. In the summarized graphs, "% (cell) activation" and CD107a, IFNγ and TNFα MFI were determined from samples co-cultured with Raji cells pretreated with the highest compound concentration (e.g., 150 μg / mL for antibodies and pAg conjugates, or 30 μg / mL or 6 μg / mL if technical issues occurred with the highest compound data).

[0356] Results / Conclusion Rituximab-conjugates activate Vδ2 γδ T cells with greater potency and efficacy than rituximab Multiple pAg conjugates conjugated with rituximab and a non-binding control were prepared at a drug-antibody ratio of approximately 2. Their binding to Raji cells was comparable to rituximab alone (Table 4), and the non-binding isotype control showed no binding. It was also determined whether the pAg conjugates were directly cytotoxic to CD20-positive Raji tumor cells. Various concentrations of pAg conjugates, ADC-XC4-r, ADC-XD4-r, ADC-XD-13-r, ADC-XS2-r, ADC-XD18-r, ADC-XC9-r, ADC-XC13-r, ADC-XS7-r, ADC-XS12-r, ADC-XS17, and ADC-XS22-r, and the respective non-binding control pAg conjugates, were incubated with Raji cells for 6 days and cell viability was determined using CellTiter-Glo®. None of the pAg conjugates tested, HMBPP and zoledronate, induced substantial (>15%) direct compound-associated cell death, whereas the positive control duocarmycin-type toxin was highly effective (Figure 7), therefore other pAg conjugates were not tested.

[0357] [Table 4]

[0358] The prepared pAg conjugates were tested for their ability to induce selective V52 γδ T cell activation after overnight incubation with Raji cells followed by 6 h of co-culture with PBMCs containing V52 γδ T cells. Dose response curves for V52 γδ T cell degranulation, IFNγ and TNFα production were generated (exemplified by CD107a production in Figure 8). These results show that Raji cells pretreated with non-binding isotype control pAg conjugates activated V52 γδ T cells to a lesser extent and EC 50 It was shown that the EC 50 and potency were calculated (Tables 5-10). Box plots show the EC 50 A summary of the efficacy, potency, and MFI are shown (Figure 9). Degranulation induced by pAg complexes correlated with IFNγ production (Figure 10). TNFα production was not assessed in all experiments, so no correlation graphs were generated for this cytokine.

[0359] The results shown in Figures 8 to 10 and Tables 5 to 10 indicate that Raji cells pretreated with pAg complexes induced the production of CD107a, IFNγ, and TNFα in a dose-dependent manner, and the production was higher in ADC-XD65-r (higher EC values ​​of CD107a, IFNγ, and TNFα) than in pretreatment with rituximab. 50 ), ADC-XD44-r (higher CD107a and IFNγ EC 50 ), and ADC-XD13-r and ADC-XS12-r (higher EC 50 ) was more potent (lower EC 50 ). All pAg conjugates, except ADC-XD65-r, activated more V52 γδ T cells (% activation) and produced greater amounts of cytokines and degranulation (MFI) than rituximab. Overall, these results showed that Raji cells preincubated with pAg conjugates potently and effectively activated V52 γδ T cells.

[0360] Most pAg conjugates were potent and effective in inducing V52 γδ T cell activity, but ADC-XD65-r was not more potent or effective than rituximab. Therefore, a DAR of 8 for this compound was generated. When pretreated with Raji cells, ADC8-XD65-r was more potent and effective than ADC-XD65-r in inducing V52 γδ T cell activation as measured by CD107a, IFNγ, and TNFα production (Figure 11). Thus, increasing the DAR leads to improved activity of weakly active pAg conjugates.

[0361] [Table 5]

[0362] [Table 6]

[0363] [Table 7]

[0364] [Table 8]

[0365] [Table 9]

[0366] [Table 10]

[0367] Example 25 Effective killing of Raji cells by V52 γδ T cells pretreated with pAg ADC-XD18-r Vδ2 γδ T cells are thought to lyse tumor cells opsonized with therapeutic antibodies such as rituximab by CD16 expressed on δ T cells (classical antibody-dependent cellular cytotoxicity, ADCC) (Sabrina Braza et al., 2011, Haematologica, 96(3), 400-407). However, not all γδ T cells express CD16 (Sabrina Braza et al, op. cit.). Vδ2 γδ T cells can also potently kill tumor cells that carry high levels of pAg. These pAg induce conformational changes in the BTN3A1 / BTN2A1 receptor complex inside the cell, activating γδ T cells and killing the target cells (Rigau et al., op. cit.). In this example, we asked whether tumor-targeting antibodies could be used as vehicles to deliver pAg to tumor cells, leading to specific tumor cell killing by Vδ2 γδ T cells. To this end, Raji cells were pretreated with rituximab-pAg conjugates and cocultured with Vδ2 γδ T cells for 1 h before examining cytotoxicity.

[0368] Materials and Methods γδ T cell proliferation To obtain large numbers of V52γδ T cells, V52γδ T cells were expanded with IL-2 and zoledronate using standard protocols (Kondo et al., 2008, Cytotherapy, 10(8):842-56. doi: 10.1080 / 14653240802419328). Frozen PBMCs (Sanquin, Nijmegen, The Netherlands) isolated from buffy coats of healthy donors were thawed and 12.5 million cells were seeded into a T25 containing 10 mL of CTS™ OpTmizer™ T cell expansion serum-free medium (CTS medium, Gibco, A3705001; basal and enriched media were premixed before use according to the manufacturer's instructions and supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco), 80 U / mL penicillin-streptomycin solution (Gibco) and Glutamax (Gibco)) supplemented with 1000 international units (IU) of recombinant human (rh) IL-2 (Miltenyi, 130-097-746) and 5 μM zoledronate (Merck) and cultured for 3 days at 37°C in a humidified incubator with 5% CO2. After 3 days, cells were transferred to T75 flasks and CTS medium supplemented with 1000 IU rhIL-2 was added. On day 8, cells were transferred to T175 flasks and CTS medium supplemented with 1000 IU rhIL-2 was added. After 13 or 14 days, cell purity and phenotype were assessed by flow cytometry. Purity of Vδ2 γδ T cells was 67.3, 81.7, 82.8, 84.5, 87, 90.6, 91.2, 92 and 95.4% relative to viable cells from 9 healthy donors used. After 14 days, killing assays were performed on expanded Vδ2 γδ T cells. For this, cells were pelleted and resuspended at 2 million cells / mL in complete growth medium (CGM; RPMI-1640 (Gibco), supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco) and 80 U / mL penicillin-streptomycin solution (Gibco)).

[0369] To determine the phenotype and purity of expanded V52 γδ T cells, 500,000 cells / well were added to a U-bottom 96-well plate, washed twice with ice-cold FACS buffer (PBS 1×, 0.1% v / w BSA, 0.02% v / v sodium azide (NaN3)) and the cell pellet was resuspended in 100 μL of the following antibody cocktail diluted in ice-cold FACS buffer: anti-V52 BV711 (clone B6, Biolegend, 1:300), anti-CD56 AlexaFluor647 (clone B159, BD Bioscience, 1:200), anti-CD16 FITC (clone 3G8, BD Biosciences, 1:50), Viability 780 (ThermoFisher Scientific, 1:1000). After 30 min incubation on ice in the dark, cells were washed twice with ice-cold FACS buffer followed by centrifugation at 300×g for 3 min and the supernatant was discarded. The pellet was resuspended in 150 μL ice-cold FACS buffer and analyzed on a BD FACSymphony A3 cell analyzer (BD Biosciences, San Jose, CA, USA). Further analysis was performed by FlowJo V10.7. Vδ2γδ T cells were defined as live Vδ2+ cells.

[0370] Killing assay Raji cells were cultured as described in Example 23.

[0371] For the killing assay, Raji cells were first washed twice with PBS (Gibco, 2326202) and then labeled with 10 μM cell proliferation dye eFluor 450 (Thermofisher Scientific, 65-0842-85) for 10 min in the dark at 37 °C. After adding 4-5 volumes of CGM for 5 min on ice, cells were washed three times with RPMI-1640 supplemented with 10% HI-FBS, diluted to 200.000 cells / mL in CGM, and 50 μL / well were seeded into 96-well plates (Greiner Bio-one, 650185, U-bottom). Various concentrations of rituximab-pAg complex, rituximab, or HMBPP at 0.1 mM or 0.013 mM diluted in CGM were then added at 50 μL per well and incubated for 16 hours in a humidified incubator at 37° C. and 5% CO2. Note that the concentration of HMBPP used was shown to induce maximum efficacy (data not shown). After 16 hours, 100 μl of CGM was added per well, cells were pelleted by centrifugation at 300×g, the supernatant was removed, and 100,000 expanded Vδ2γδ T cells (culture day 14) were added at 50 μL per well to each well. The plates were incubated for 1 hour in a humidified incubator at 37° C. and 5% CO2. Cells were then pelleted by centrifugation at 300×g for 3 minutes and the supernatant was removed. Cells were resuspended in 50 μL of Viability Stain 780 (BD Biosciences, diluted in 1000× ice-cold FACS buffer) plus anti-CD19 FITC (Miltenyi 130-113-645), incubated on ice in the dark for 30 min, and cells were washed by adding 150 μL of ice-cold FACS buffer and centrifugation at 300×g for 3 min. The cell pellet was then resuspended in 50 μL of BD cytofix solution (554655) and incubated on ice in the dark for 15 min, followed by washing twice with 150 μL of ice-cold FACS buffer and centrifugation (300×g, 3 min) to remove the supernatant. Finally, cells were resuspended in 150 μL of ice-cold FACS buffer and analyzed on a BD FACSymphony A3 cell analyzer (BD Biosciences, San Jose, CA, USA).Raji cells were gated using eFluor450 dye, and the percentage of dead Raji cells was determined by viability staining and further analyzed by FlowJo V10.7. Efficacy = % of dead Raji cells pretreated with highest concentration of compound - % of dead Raji cells pretreated without compound

[0372] Results / Conclusion When Raji cells were pretreated with rituximab, dose-dependent killing was detected by expanded V52 γδ T cells from most donors (Figure 12). Rituximab potency was variable and correlated, as expected, with CD16 expression on V52 γδ T cells, and varied widely between donors (Figure 12B). Rituximab pretreatment induced consistently lower potency compared to HMBPP pretreatment. When V52 γδ T cells were exposed to ADC-XD18-r pretreated Raji cells, dose-dependent killing of Raji cells was observed, with potency in a similar range to HMBPP pretreated Raji cells and higher than rituximab (Figures 12A and 12D). The activity of unbound isotype pAg complexes was low, indicating a reliable EC for V52 γδ T cell activation. 50 was not possible to calculate (Figures 12A and 12C). Overall, these results demonstrate that rituximab-pAg conjugates can potently and effectively target tumor cells for destruction by V52 γδ T cells in a TAA-specific manner.

[0373] Example 26 CD20 positive cell lines derived from various B cell malignancies potently and efficiently activate V52 γδ T cells after preincubation with ADC-XD18-r The activity of ADC-XD18-r was tested with multiple CD20-positive cell lines representing B cell malignancies (CLL, NHL) with different levels of CD20 expression (Table 11). For this, the B cell lines were first pretreated with compounds for 16 hours and then co-cultured with PBMCs. Activation of Vδ2γδ T cells was assessed by determining the level of degranulation (CD107a production).

[0374] Materials and Methods Functional assays Raji cells were cultured as described in Example 23. Human tumor cell lines MEC-1, HG-3, SU-DHL-4 and SU-DHL-8 cells were obtained from the German collection of Microorganisms and cell cultures GmbH (DSMZ, Leibniz Institute, Germany). HG-3 and SU-DHL-4 cells were cultured in CGM. SU-DHL-8 were cultured in RPMI-1640 (Lonza) supplemented with 20% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco) and 80 U / mL penicillin-streptomycin solution (Lonza). MEC-1 cells were cultured in IMDM (12-722F, IMDM, Lonza) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco- Life Technologies; Carlsbad, CA) and 80 U / mL penicillin-streptomycin solution (Lonza Group Ltd, Basel Switzerland). All cells were maintained at 37°C in a humidified incubator with 5% CO2 and passaged twice weekly.

[0375] For materials and methods of functional assays, see Example 23, except that only degranulation levels were determined, not IFNγ expression levels. Therefore, on day 3 of the experiment, cells were directly analyzed on a BD FACSymphony A3 cell analyzer (BD Biosciences, San Jose, CA, USA) before incubation with BD Perm / wash. The highest compound concentrations used to pretreat Raji cells were 150 μg / mL for antibodies / ADCs and 0.1 mM for HMBPP. EC 50was calculated in GraphPad Prism as the concentration (μg / mL) midway between the bottom and top of the curve response. In the summarized graphs, "% Vδ2 γδ T cell activation" was determined from samples co-cultured with Raji cells pre-treated with the highest compound concentration (e.g. 150 μg / mL for antibodies and ADCs).

[0376] Determination of CD20 expression levels The expression level of CD20 receptor was determined using a human calibrator kit (Biocytex, CP010). Target cells (100,000 cells / well in a 96-well plate) were washed twice with ice-cold FACS buffer (PBS+0.1% v / w BSA+0.02% v / v sodium azide (NaN3)) followed by the addition of 50 μL per well of various concentrations of rituximab (anti-CD20) diluted in ice-cold FACS buffer. After 30 min of incubation at 4° C., cells were washed twice with ice-cold FACS buffer and resuspended in 50 μL of FACS buffer. Then, 50 μL of beads from the human calibrator kit were added to separate wells of the 96-well plate. A two-fold concentrated stock of APC-conjugated secondary F(ab')2 goat anti-human IgG (Fc fragment specific, Jackson ImmunoResearch, 1:3000, final 1:6000) was made and added to the cells and beads at 50 μL / well. After 30 min incubation at 4°C in the dark, the cells and beads were washed twice in ice-cold FACS buffer and resuspended in 150 μL ice-cold FACS buffer. Fluorescence intensity was determined by flow cytometry using a FACSymphony (BD Biosciences) and absolute receptor numbers were determined according to the manufacturer's instructions. Experiments were performed in duplicate.

[0377] Results / Conclusion The B cell lines used in this example expressed CD20 at various levels (Table 11).

[0378] [Table 11]

[0379] All B cell lines tested had the capacity to activate V52 γδ T cells, as HMBPP pretreatment induced degranulation of V52 γδ T cells (Figure 13A). When B cell lines were pretreated with various concentrations of ADC-XD18-r, they all induced strong activation of V52 γδ T cells with higher potency (% activation) and potency than rituximab itself (Figures 13B-13F, Table 11). Non-binding control ADC induced V52 γδ T cell activation with low / negligible potency and efficacy. These results showed that ADC-XD18-r activated V52 γδ T cells when preincubated with multiple B cell malignancies with low / high expression levels of CD20, whereas pretreatment with non-binding pAg complexes failed to activate V52 γδ T cells.

[0380] Example 27 HER2 pretreated with trastuzumab-ADC high Cells induce Vδ2 γδ T cell activation To demonstrate activity of the pAg conjugates over rituximab, the linker-drug XD18 was conjugated with trastuzumab to produce ADC-XD18-t. V52 γδ T cell activity was determined upon pretreatment of HER2-positive cell lines (listed in Table 12) with these new ADCs and co-incubation with PBMCs.

[0381] Materials and Methods Functional assays Functional assays were performed as disclosed in Example 23. In addition to anti-IFNγ BV650, anti-TNFα PE (BD Biosciences, San Jose, CA, USA, clone Mab11) was also included in the intracellular cytokine staining step in most experiments. The highest compound concentrations used in the pretreatment of Raji cells were 150 μg / mL for antibodies / ADCs and 0.1 mM for HMBPP. EC 50was calculated in GraphPad Prism as the concentration (μg / mL) midway between the bottom and top of the curve response. In the summarized graphs, "% Vδ2 γδ T cell activation" was determined from samples co-cultured with Raji cells pre-treated with the highest compound concentration (e.g., 150 μg / mL for antibody and pAg complexes).

[0382] Human tumor cell lines SK-BR-3, BT-474, and SK-OV-3 were obtained from the American Type Culture Collection (ATCC, Rockville, MD), and HCT-116 from the German collection of Microorganisms and cell cultures GmbH (DSMZ, Leibniz Institute, Germany). BT-474 (ATCC; ATCC-HTB-20) was cultured in CGM and maintained at 37°C in a humidified incubator with 5% CO2 and passaged twice weekly. SK-BR-3, SK-OV-3, and HCT-116 were maintained in McCoys 5A medium (Lonza) containing 10% v / w FBS HI and 80 U / mL penicillin-streptomycin solution (Lonza).

[0383] Determination of HER2 expression levels Trastuzumab was used to determine HER2 levels on cells, see Example 25. Experiments were performed in duplicate and the average sABC is reported.

[0384] Results / Conclusion These cell lines expressed high (BT-474, SK-BR-3, and SK-OV-3) or low (HCT-116) levels of HER2 (Table 12).

[0385] [Table 12]

[0386] Four different cell lines were first pre-incubated with ADC-XD18-t, ADC-XD18-i or trastuzumab and then co-cultured with PBMCs containing V52 γδ T cells to determine immune cell activation. Results from a representative donor are shown in Figure 14 and show potency (EC 50) and efficacy are summarized in Figure 15. BT-474, SK-BR-3 and SK-OV-3 cells preincubated with trastuzumab induced activation of V52 γδ T cells in a dose-dependent manner, likely via trastuzumab-FcγR interactions. HCT-116 cells pretreated with trastuzumab failed to significantly activate V52 γδ T cells, likely due to the low number of HER2 receptors on the cell surface of HCT-116 cells (Table 12). When preincubated with BT-474, SK-BR-3 and SK-OV-3 cells, ADC-XD18-t induced degranulation (CD107a), IFNγ and TNFα production in V52 γδ T cells more than trastuzumab itself. HCT-116 cells failed to activate V52 γδ T cells when preincubated with ADC-XD18-t. This was not due to a lack of ability of HCT-116 cells to activate V52 γδ T cells, as HCT-116 pretreated with HMBPP were able to activate V52 γδ T cells (Figure 16). Pretreatment of BT-474, SK-BR-3, SK-OV-3 and HCT-116 cells with non-binding isotype control-ADC resulted in negligible or low activation of V52 γδ T cells (illustrated in Figure 14). Trastuzumab pretreated BT-474, SK-BR-3 and SK-OV-3 also activated NK cells, likely via FcγRs that are well known to be expressed on NK cells. Trastuzumab pAg conjugate pretreated BT-474, SK-BR-3 and SK-OV-3 cells were also able to activate NK cells to a similar extent (Figure 17), indicating that trastuzumab-induced effector functions were not compromised after addition of pAg conjugates. HCT-116 cells did not significantly activate NK cells after preincubation with trastuzumab or trastuzumab-ADC, again indicating that HER2 expression levels were too low in this cell line to activate immune cells. Overall, these results indicate that pAg complexes can induce Vδ2 γδ T cell activation in combination with a variety of tumor-targeting antibodies that target multiple malignancies.

[0387] Example 28 High pAg drug-antibody ratio (DAR) improves V52 γδ T cell activity against cells with low expression of tumor-associated antigens (TAA) We investigated whether increasing the DAR would potently and efficiently activate Vδ2 γδ T cells, particularly in TAA-underrepresented cell lines.

[0388] Materials and Methods Vδ2 γδ T cell activation assay Functional assays Functional assays were performed as disclosed in Example 23, except that Raji and MOLM-13 cells were pretreated with antibody / ADC / control for 16 or 40 hours (Table 13). Furthermore, only the level of CD107a degranulation was determined, not the level of IFNγ expression. Therefore, on day 3 of the experiment, cells were analyzed directly on a BD FACSymphony A3 cell analyzer (BD Biosciences, San Jose, CA, USA) before incubation with BD Perm / wash. The highest compound concentrations used to pretreat Raji and MOLM-13 cells were 150 μg / mL for antibody / ADC and 0.1 mM for HMBPP. EC 50 was calculated in GraphPad Prism as the concentration (μg / mL) midway between the bottom and top of the curve response. In the summarized graphs, "% Vδ2 γδ T cell activation" was determined from samples co-cultured with Raji or MOLM-13 cells pre-treated with the highest compound concentration (e.g., 150 μg / mL for antibodies and ADCs).

[0389] [Table 13]

[0390] Determination of CD123 expression levels Expression levels of CD123 receptor were determined using the Qifi kit (DAKO, Agilent, USA). Cells (100,000 cells / well in 96-well plates) were washed twice with ice-cold FACS buffer (PBS+0.1% v / w BSA+0.02% v / v sodium azide (NaN3)) followed by the addition of 50 μL per well of various concentrations of anti-CD123 (clone 6H6, ThermoFisher Scientific) diluted in ice-cold FACS buffer. After 30 min incubation at 4°C, cells were washed twice with ice-cold FACS buffer and resuspended in 50 μL of APC-complexed secondary F(ab')2 goat anti-human IgG (Fc fragment specific, Jackson ImmunoResearch, final 1:6000) in ice-cold FACS buffer. Dilutions of secondary antibody were also added to the pelleted beads from the Qifi kit. Incubation with secondary antibodies was performed in the dark on ice at 4° C. for 30 min, after which cells and beads were washed twice with ice-cold FACS buffer and resuspended in 150 μL ice-cold FACS buffer. Fluorescence intensity was determined by flow cytometry using a FACSVerse or FACSymphony (BD Biosciences) and absolute receptor numbers were determined according to the manufacturer's instructions.

[0391] MOLM-13 and Raji cell cultures Raji cells were cultured as described in Example 23. The CD123-positive acute monocytic leukemia cell line MOLM-13 (DSMZ, ACC 554, the German collection of Microorganisms and cell cultures GmbH (Leibniz Institute, Germany)) was cultured in CGM, maintained at 37° C. in a humidified incubator with 5% CO2, and passaged twice a week.

[0392] Results / Conclusion The MOLM-13 cell line expressed low levels of CD123 (Table 14). CD20 expression levels of Raji cells are shown in Table 12.

[0393] [Table 14]

[0394] Both MOLM-13 and Raji cells were able to activate V52 γδ T cells after pretreatment with HMBPP (Figure 18A). The prepared pAg conjugates and control compounds were tested for their ability to induce selective V52 γδ T cell activation after overnight incubation with MOLM-13 or Raji cells followed by 6 h of co-culture with PBMCs containing V52 γδ T cells. A dose-response curve for V52 γδ T cell degranulation was generated (Figures 18B-18E). These results showed that cells pretreated with non-binding isotype control pAg conjugates activated V52 γδ T cells to a lesser extent. Thus, EC 50 Values ​​could not be reliably calculated. Rituximab / anti-CD123 41C For MoAb pAg complexes, EC 50 , potency and MFI were calculated (Figures 18F-H).

[0395] anti-CD123 41C When MoAb-pretreated MOLM-13 cells were cocultured with PBMCs, Vδ2 γδ T cell activation was not measured in three of four donors. 41C When MOLM-13 cells pretreated with pAg were co-cultured with PBMCs, the percentage of activated V52 γδ T cells was increased. In addition, the amount of CD107a produced per cell was higher with the pAg complex compared to mAb alone (Fig. 18H). Increasing the DAR led to a further increase in the percentage of CD107a-positive V52 γδ T cells (Fig. 18G, CD123 41CADC). Higher DAR did not improve the potency of anti-CD20-based ADCs (Figure 4G), likely because maximal activity was already reached at DAR2. The potency of anti-CD20 ADCs improved with higher DAR, but so did that of the non-binding isotype control. Overall, these results indicate that anti-CD123 pAg conjugates are better able to activate V52 γδ T cells compared to mAb alone, and that higher DAR improves the potency of anti-CD123 pAg conjugates.

Claims

1. A complex comprising a targeting moiety covalently linked to an immunomodulatory moiety, wherein the immunomodulatory moiety is a phosphoantigen.

2. The complex according to claim 1, wherein the targeting moiety is a tumor targeting antibody or an antigen-binding fragment thereof.

3. The complex according to claim 1, wherein the phosphoantigen is pyrophosphate, pyrophosphonate, bisphosphonate, monophosphate or monophosphonate or a prodrug thereof, and comprises an allyl alcohol group.

4. The complex according to claim 1, wherein the targeting moiety is bound to the phosphoantigen via a linking moiety.

5. Formula I: Tm-(L-(pAg) ｘ ) ｙ (I) (wherein, Tm represents a targeting moiety, L represents a linking moiety, pAg represents a phosphoantigen, x represents the number of phosphoantigens per linking moiety and is an integer from 1 to 5, y represents the number of L-(pAg) per Tm (targeting moiety) and is from 1 to 10) ｘ ) The complex according to claim 1, represented by.

6. The complex according to claim 5, wherein y is from 1 to 8.

7. The complex according to claim 4, wherein the linking moiety comprises a cleavable linker.

8. Formula (II): 【Chemical 1】 (wherein, Q is a structure represented by Formula IIa or Formula IIb: 【Chemical 2】 ; Y is halogen; W １ is N, CH or CF, preferably CH; W ２ is CH ２ , CHF, CF ２ or O; X １ is O, S, NH, CH ２ , CHF or CF ２ ; X ２ is O, CH ２ , CHF or CF ２ ; X ３ is absent or is O or NH; X ４ａ～ｄ is independently selected from O and S, respectively; X ５ is CH ３ , CH ２ F, CHF ２ , CF ３ or CCl ３ ; x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R １ is H, or a connecting part with the linking part (L) or the prodrug part; R ２ is a connection to H, or to the linking moiety (L), Cat+ or the prodrug moiety; R ３ is H, or a connecting part to the linking moiety (L), Cat+ or a prodrug moiety; R ４ is H, or a connecting part with a linking part (L), Cat+ or a prodrug part; When n is 0, R ３ and R ２ are connected by a C １～６ (hetero)alkyl group; When n is 1 or 2, R ３ and R ４ are connected by a C １～６ (hetero)alkyl group) A linker-drug compound comprising one or more phosphoantigens covalently linked to a linking moiety (L) having the structure represented by.

9. The linker-drug compound according to claim 8, wherein Q is a structure represented by Formula IIa.

10. W １ is CH, and X ５ is CH ３ and R １ is H or a connecting portion with the linking moiety (L), the linker-drug compound according to claim 9.

11. X ３ is O, and R ３ is a connection to a cleavable linking moiety, and R １ is H, the linker-drug compound of claim 9.

12. W ２ is CH ２ The linker-drug compound according to claim 8, wherein W is CH and m is 1.

13. X １ is CH ２ The linker-drug compound according to claim 8, wherein

14. X ３ is O, R ３ is a connection to a cleavable linking moiety, W １ is CH, X ５ is CH ３ and R １ is H, W ２ is CH ２ and m is 1, X １ is CH ２ A linker-drug compound according to claim 9, wherein

15. n is 0 or 1, and X ４ａ～ｂ and X ４ｃ～ｄ (when present) is O, R ２ and R ４ (when present) is H, the linker-drug compound according to claim 8.

16. R ２ and R ３ are - Pivaloyloxymethyl (POM) group and isopropyloxycarbonyloxymethyl (POC) group, - Substituted or unsubstituted (hetero)aryl group, and - Formula IV or Formula V: [Chemical 3] (wherein; R ａ and R ａ’ are independently selected from H, an optionally substituted amino acid side chain, and an apolar side chain containing an optionally substituted C １～１４ alkyl chain, R ｂ is H, benzyl or substituted or unsubstituted (C １ to ８ ) alkyl, R ｃ and R ｃ’ are each independently selected from H, and optionally substituted, C １ -C ６ alkyl, C ３ -C ６ cycloalkyl, aryl or heteroaryl). The structure represented by, A prodrug moiety selected from the group consisting of.

17. R ２ and R ３ The linker-drug compound according to claim 16, wherein R and R are independently selected from a POM group and a POC group.

18. n is 0, and R ２ is a substituted or unsubstituted 5- or 6-membered (hetero)aryl group, and R ３ has the structure represented by Formula IV or Formula V, or vice versa, the linker-drug compound according to claim 16.

19. The linking moiety (L) is of Formula VI or Formula VII: 【Chemical 4】 (wherein, m is an integer from 1 to 10, preferably 5; AA is an amino acid, preferably a natural amino acid; p is 0, 1, 2, 3 or 4; q is an integer from 1 to 12, preferably 2; ES is absent or is an extended spacer selected from the following: 【Chemical Formula 5】 (wherein, R ４ is H, halogen, CF ３ , C １～４ alkyl, C ２～４ alkenyl, C ２～４ alkynyl, C １～４ alkoxyl or C １～４ alkylthio, preferably H, F, CH ３ , CF ３ , more preferably H or F; V is H, ethyl, -(CH ２ CH ２ O) ｐ -OMe, CH ２ CH ２ SO ２ Me or CH ２ CH ２ N(Me) ２ and is; p is an integer from 1 to 12)) The linker-drug compound according to claim 8, comprising the structure shown by

20. The following structure [Chemical Formula 6] The linker-drug compound according to claim 8, having

21. Use of the linker-drug compound according to any one of claims 8 to 20 in the manufacture of the conjugate according to any one of claims 1 to 7.

22. A conjugate comprising a tumor-targeting antibody or an antigen-binding fragment thereof conjugated with the linker-drug compound according to any one of claims 8 to 20.

23. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 7 and one or more pharmaceutical additives.

24. The pharmaceutical composition according to claim 23, for treating cancer, an autoimmune disease or an infectious disease.

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