PEPTIDE DERIVATIVES AND CONJUGATES THEREOF FOR TREATING CANCER

Abstract

Provided herein are luteinizing hormone-releasing hormone (LHRH) peptide derivatives that target the LHRH receptor. LHRH peptide derivatives, LHRH peptide-drug conjugates (LHRH-PDCs) and methods of using the derivatives and/or conjugates thereof to treat a LHRH receptor expressing cancer are described.

Claims

1. A LHRH peptide-drug conjugate (LHRH-PDC) comprising the sequence:
X.sub.1-His-Trp-Ser-X.sub.2-X.sub.3(L-D)-X.sub.4-X.sub.5-Pro-NHR wherein X.sub.1 is pGlu or Gln; X.sub.2 is Tyr, Phe or His; X.sub.3 is D-Lys or D-Lys(Ahx); X.sub.4 is Leu, Val, Trp or Met; X.sub.5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; R is CH.sub.2CH.sub.3 or CH.sub.3; wherein L is a linker; and D is a cytotoxic agent, or a pharmaceutically acceptable salt thereof.

2.-4. (canceled)

5. The LHRH-PDC of claim 1, wherein X.sub.1 is Gln, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3.

6. The LHRH-PDC of claim 1, wherein X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3.

7. The LHRH-PDC of claim 1, wherein X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys(Ahx), X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3.

8.-20. (canceled)

21. The LHRH-PDC of claim 1, wherein the linker is a self-immolative linker.

22. The LHRH-PDC of claim 1, wherein the linker is maleimidocaproyl valine-citrulline-p-aminobenzyl carbamoyl (mc-vc-PABC).

23. The LHRH-PDC of claim 1, wherein the cytotoxic agent is an anti-mitotic agent, an alkylating agent, an anti-metabolite, a topoisomerase inhibitor or a protein kinase inhibitor.

24. The LHRH-PDC of claim 1, wherein the cytotoxic agent is a vinca alkaloid, a cryptophycin, bortezomib, thiobortezomib, a tubulysin, aminopterin, rapamycin, paclitaxel, docetaxel, daunorubicin, everolimus, a-amanatin, vemcarin, didemnin B, geldanomycin, purvalanol A, ispinesib, budesonide, dasatinib, an epothilone, a maytansine, doxorubicin, camptothecin, methotrexate (MTX) or monomethyl auristatin E (MMAE).

25. The LHRH-PDC of claim 1, wherein the cytotoxic agent is MMAE.

26. An anti-proliferative LHRH peptide derivative comprising the sequence:
X.sub.1-His-Trp-Ser-X.sub.2-X.sub.3-X.sub.4-X.sub.5-Pro-NHR wherein X.sub.1 is pGlu or Gln; X.sub.2 is Tyr, Phe or His; X.sub.3 is D-Lys or D-Lys(Ahx); X.sub.4 is Leu, Val, Trp or Met; X.sub.5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; and R is CH.sub.2CH.sub.3 or CH.sub.3, or a pharmaceutically acceptable salt thereof: wherein X.sub.1 is Gln, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3; X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3: or X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys(Ahx), X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3.

27.-54. (canceled)

55. The anti-proliferative LHRH peptide derivative of claim 26, wherein the LHRH peptide derivative is conjugated to a cytotoxic agent via a self immolative linker.

56.-60. (canceled)

61. A pharmaceutical composition comprising a LHRH-PDC of claim 1 or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient.

62. A method of treating a cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 61, wherein the subject has a LHRH receptor expressing cancer.

63. The method of claim 62, wherein the subject has a LHRH receptor expressing cancer selected from the group consisting of breast cancer, a hormone sensitive and/or refractory breast cancer, prostate cancer, colon cancer, ovarian cancer, pancreatic cancer and endometrial cancer.

64. The method of claim 62, wherein the subject has a LHRH receptor expressing cancer, wherein the LHRH receptor expressing cancer is Triple Negative Breast Cancer (TNBC).

65.-73. (canceled)

74. The method of claim 62, wherein: X.sub.1 is Gln, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3; X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3; or X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys(Ahx), X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3.

75. The method of claim 62, wherein the cytotoxic agent is MMAE LHRH-PDC and the linker is mc-vc-PABC.

76. The LHRH-PDC of claim 1, comprising the sequence Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 or a pharmaceutically acceptable salt thereof, wherein the cytotoxic agent is MMAE and the linker is maleimidocaproyl valine-citrulline-p-aminobenzyl carbamoyl (mc-vc-PABC), optionally with at least one pharmaceutically acceptable excipient.

77. The LHRH-PDC of claim 1, comprising the sequence pGlu-His-Trp-Ser-Tyr-D Lys-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof, wherein the cytotoxic agent is MMAE and the linker is maleimidocaproyl valine-citrulline-p-aminobenzyl carbamoyl (mc-vc-PABC), optionally with at least one pharmaceutically acceptable excipient.

78. The LHRH-PDC of claim 1, comprising the sequence pGlu-His-Trp-Ser-Tyr-D Lys(Ahx)-Leu-Arg-Pro-NHCH2CH3 or a pharmaceutically acceptable salt thereof, wherein the cytotoxic agent is MMAE and the linker is maleimidocaproyl valine-citrulline-p-aminobenzyl carbamoyl (mc-vc-PABC), optionally with at least one pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0079] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings as follows.

[0080] FIGS. 1A and 1B: (A) Structure of exemplary LHRH peptide derivatives: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 (LD4), pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 (LD5) and pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 (LD6). With the exception of D-Lys at the sixth position, all amino acids in the LHRH peptide derivatives of FIG. 1A are present in their L-form. (B) The metabolic stability of LHRH peptide derivatives examined in human plasma and quantified by Liquid Chromatography-Mass-Spectrometry (LC-MS) in a time dependent manner. The LHRH peptide derivatives of the invention exhibit substantially improved half-life (T.sub.1/2 of about 257 min, 365 min and 309 min for LD4, LD5 and LD6, respectively) compared to the native LHRH peptide (T.sub.1/2 of about 10 min) and LHRH agonists triptorelin ([w.sup.6] LHRH) and [k.sup.6] LHRH (T.sub.1/2 of about 19 min and 39 min, respectively). Statistical analysis was performed using a one-way ANOVA followed by Dunnett's post-hoc test. Error bars are standard deviations (STDV), p<0.05, n=9 (3 independent experiments each performed in triplicate).

[0081] FIG. 2: LHRH receptor (LHRH-R) expression level in 3 breast cancer cell lines (MCF-7, MDA-MB-231 and SK-BR-3) versus 3-actin as the control determined using Western blot analysis. The breast cancer cell lines have different characteristics: MDA-MB-231: ER−, HER-2−, PR−; MCF-7: ER+, HER-2+, PR+; and SK-BR-3: ER−, HER-2+, PR−. A major protein band of approximately 64 kDa molecular mass known for the human pituitary LHRH receptor was identified in MCF-7, MDA-MB-231 and SK-BR-3 (lanes 1, 2, and 3 respectively). The level of LHRH-R expression relative to β-actin in MCF-7, MDA-MB-231 and SK-BR-3 showed that these breast cancer cells can be actively targeted through the LHRH receptor. The Western blot analysis was performed using the antibody specifically raised against the LHRH-R.

[0082] FIGS. 3A and 3B: (A) The expression of LHRH-Rs was further confirmed by immunohistochemistry in MDA-MB-231 (TNBC cell model), SKOV-3 (low LHRH-R expressing cell model), HMEC and MCF-10A as normal breast cells. FIG. 3A shows specific receptor binding of primary antibody labelled by Rhodamine-conjugated secondary antibody. (B) The LHRH-R signal intensity was quantified by Fiji J software in normal breast cells (HMEC and MCF-10A), MDA-MB-231 and SKOV-3. The signal intensity in these cell lines were assessed by One-way ANOVA followed by Tukey's multiple comparisons test (****p<0.0001). The LHRH-R expression signal intensity is significantly higher in MDA-MB-231 compared to SKOV-3, HMEC and MCF-10A. Error bars are standard deviations (STDV), n=9 (3 independent experiments each performed in triplicate).

[0083] FIG. 4A to 4E: The cytotoxic activity of the peptide conjugate LD5-mc-vc-PABC-MMAE (LM) in comparison to MMAE was assessed in MDA-MB-231 (TNBC cell model) versus HMEC and MCF-10A (normal breast cells) and SKOV-3 (low LHRH-R expressing cell model), by MTT assay after incubating cells with compounds in serial dilution for 72 h. (A) The relative cellular viability was assessed in comparison to PBS-treated negative control group and IC.sub.50 value (nM) was calculated by non-linear regression (curve fit) for HMEC and MCF-10A (normal breast cells) and MDA-MB-231 (LHRH-R positive, TNBC cell model) and SKOV-3 (LHRH-R low expressing cells). (B) The cell viability in LD5-me-vc-PABC-MMAE (LM) was assessed in HMEC as normal breast cell line in comparison to MMAE by two-way ANOVA followed by Sidak's multiple comparisons test (****p<0.0001). (C) The cell viability in LD5-mc-vc-PABC-MMAE (LM) was assessed in MCF-10A as normal breast cell line in comparison to MMAE by two-way ANOVA followed by Sidak's multiple comparisons test (***p<0.001, **p<0.01, *p<0.05). (D) The cell viability in LD5-me-vc-PABC-MMAE (LM) was assessed in MDA-MB-231 (TNBC cell model) in comparison to MMAE by two-way ANOVA followed by Sidak's multiple comparisons test (****p<0.0001). (E) The cell viability in LD5-me-vc-PABC-MMAE (LM) was assessed in SKOV-3 (LHRH-R low expressing cells) in comparison to MMAE by two-way ANOVA followed by Sidak's multiple comparisons test (****p<0.0001). LD5-mc-vc-PABC-MMAE (LM) conjugate shows decreased cytotoxicity compared to MMAE due to selectivity of LHRH uptake via the receptor rather than simple diffusion of free MMAE in the cell lines. Error bars are standard deviations (STDV), n=9 (3 independent experiments each performed in triplicate).

[0084] FIGS. 5A and 5B: Receptor binding competitive assay. MDA-MB-231 was pre-treated with triptorelin (TRN) and LM (LD5-mc-vc-PABC-MMAE) then the MTT assay was performed. (A) The IC.sub.50 value (nM) was calculated by non-linear regression (curve fit) for PC-3 and MDA-MB-231 with and without pre-treatment with triptorelin. (B) The cell viability in TRN-LM was assessed in comparison to LM in MDA-MB-231 (TNBC cell line). The cytotoxic effects of LM is reversed after pre-treatment with triptorelin indicating a significant role of LHRH-Rs in the mechanism of action of LM. Two-way ANOVA was performed followed by Sidak's multiple comparisons test (***p<0.00001). Error bars are standard deviations (STDV), n=8 (3 independent repeats).

[0085] FIG. 6: Proximity ligation assay was used to show the protein-protein interaction between LHRH-R and LHRH in MDA-MB-231 (TNBC cell model) treated with LD5-me-vc-PABC-MMAE (1 μM) compared to negative control (PBS). Stained nucleus (DAPI blue) and PLA signal (TexasRed) were detected by LEICA SPE2 confocal LSM.

[0086] FIGS. 7A and 7B: In vitro uptake study of LD5-me-vc-PABC-MMAE in TNBC cell line and normal breast cells. (A) MDA-MB-231 as a TNBC cell model, MCF-10A and HMEC as normal breast cells were treated with 1 μM of LD5-me-vc-PABC-MMAE (LM) for 18 h. LM was stained using monoclonal primary antibody specific for LHRH and secondary antibody conjugated with Rhodamine and detected by LEICA SPE2 confocal LSM. (B) The LM signal intensity was measured by Fiji J software in normal breast cells (HMEC and MCF-10A) and TNBC cell model (MDA-MB-231). There was significantly higher uptake of LM in LHRH-R positive cells (MDA-MB-231) compared to normal breast cells. The signal intensity in these cell lines were assessed by one-way ANOVA followed by Tukey's multiple comparisons test (****p<0.0001). Error bars are standard deviations (STDV), n=8 (3 independent repeats).

[0087] FIGS. 8A and 8B: The effect of MMAE and LD5-me-vc-PABC-MMAE (LM) on α-tubulin polymerization. (A) MDA-MB-231 (TNBC cell model) and normal breast cells (HMEC and MCF-10A) were treated with free MMAE and LM at 1 μM and PBS (control) followed by immunostaining of α-tubulin with primary and secondary antibodies. The α-tubulin signals were detected by LEICA SPE2 confocal LSM. (B) The α-tubulin signal intensity was measured by Fiji J software in normal breast cells (HMEC and MCF-10A) and MDA-MB-231. ****p<0.0001 is the significance in the signal intensity in each cell line treated with PBS and LM in comparison to MMAE-treated cells. ####p<0.0001 is the significance of the α-tubulin signal intensity in MDA-MB-231 cells treated with LM in comparison to MCF-10A and HMEC cells. The α-tubulin signal in normal breast cells treated with PBS and LM were significantly higher than their MMAE treated counterpart indicating the lower impact of the targeted drug on the α-tubulin polymerization as a factor of cell survival. Statistical analysis was made in Prism by two-way ANOVA followed by Tukey's multiple comparisons test. Error bars are standard deviations (STDV), n=8 (3 independent repeats).

[0088] FIGS. 9A and 9B: (A) Cells were co-transfected with LHRH-R siRNAs and fluorescent labelled siRNA negative control as transfection efficiency signal (FAM-transfection control). Negative control refers to MDA-MB-231 cells that were not transfected with LHRH-R siRNAs and FAM-transfection control. LHRH-R expression was detected by immunocytochemistry with specific receptor binding of primary antibody labelled by Rhodamine-conjugated secondary antibody. Immunocytochemical images were captured by LEICA SPE2 confocal LSM. (B) The LHRH-R signal intensity in both silenced LHRH-R and negative control was measured by Fiji J software. Silencing of LHRH-Rs results in significantly reduced LHRH-R expression in transfected MDA-MB-231 cells compared to negative control. The data was analysed by unpaired t-test with Welch's correction comparison between the silenced LHRH-R and negative control (**** p<0.0001). Error bars are standard deviations (STDV), n=8 (3 independent repeats).

[0089] FIGS. 10A and 10B: Uptake of LD5-me-vc-PABC-MMAE (LM) after silencing LHRH-R in MDA-MB-231 cells. The cells were treated with LHRH-R siRNA and co-transfected with fluorescent labelled siRNA negative control as transfection efficiency signal (FAM-transfection control). LHRH-R negative control refers to MDA-MB-231 cells that were not transfected with LHRH-R siRNA and FAM-transfection control. (A) Silenced LHRH-R cells and negative control cells were treated with LM (1 μM) for 18 h and LM signal was detected by immunocytochemistry with specific LHRH binding of primary antibody labelled by Rhodamine-conjugated secondary antibody. Immunocytochemical images were captured by LEICA SPE2 confocal LSM. (B) The LM signal intensity in both silenced LHRH-R and negative control was measured by Fiji J software. The uptake of LD5-me-vc-PABC-MMAE was significantly reduced after silencing the LHRH-R gene indicating that the construct is actively targeted to and up-taken by cancer cells via overexpressed LHRH-Rs. The data was analysed by unpaired t-test with Welch's correction (**** p<0.0001). Error bars are standard deviations (STDV), n=8 (3 independent repeats).

[0090] FIG. 11: Stability of LHRHD-(mc-vc-PABC)-MMAE (i.e. LD5-mc-vc-PABC-MMAE) in human plasma, mouse plasma, and cell culture media (c-media). The stability was measured based on the presence of free MMAE detected in the samples by LC-MS at each time point relative to time 0. Error Bars are standard deviations (STDV), p>0.05 and n=9 (mean is calculated from samples in triplicate in 3 independent experiments).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0091] In one embodiment, provided is a LHRH-PDC comprising the sequence:

X.sub.1-His-Trp-Ser-X.sub.2-X.sub.3(L-D)-X.sub.4-X.sub.5-Pro-NHR [0092] wherein [0093] X.sub.1 is pGlu or Gln; [0094] X.sub.2 is Tyr, Phe or His; [0095] X.sub.3 is D-Lys or D-Lys(Ahx); [0096] X.sub.4 is Leu, Val, Trp or Met; [0097] X.sub.5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; [0098] R is CH.sub.2CH.sub.3 or CH.sub.3; [0099] wherein [0100] L is a linker; and [0101] D is a cytotoxic agent,
or a pharmaceutically acceptable salt thereof. In certain embodiments, X.sub.1 is Gln, X.sub.3 is D-Lys and the R is CH.sub.2CH.sub.3. In some embodiments, X.sub.1 is pGlu, X.sub.3 is D-Lys and R is CH.sub.2CH.sub.3. In some embodiments, X.sub.1 is pGlu, X.sub.3 is D-Lys(Ahx) and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.1 is Gln, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys(Ahx), X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, the linker is a cleavable linker. In some embodiments, the linker is an uncleavable linker. In some embodiments, the cleavable linker is a self-immolative linker. In a related embodiment, the self-immolative linker is maleimidocaproyl valine-citrulline-p-aminobenzyl carbamoyl (mc-vc-PABC). In certain embodiments, the cytotoxic agent is an anti-mitotic agent, an alkylating agent, an anti-metabolite, a topoisomerase inhibitor or a protein kinase inhibitor. In some embodiments, the cytotoxic agent is selected from the group consisting of a vinca alkaloid, a cryptophycin, bortezomib, thiobortezomib, a tubulysin, aminopterin, rapamycin, paclitaxel, docetaxel, daunorubicin, everolimus, a-amanatin, vemcarin, didemnin B, geldanomycin, purvalanol A, ispinesib, budesonide, dasatinib, an epothilone, a maytansine, doxorubicin, camptothecin, methotrexate (MTX) or monomethyl auristatin E (MMAE). In certain embodiments, the cytotoxic agent is MMAE.

[0102] In some embodiments, the linker comprises at least one amino acid. In certain embodiments, the linker comprises one or more amino acid residues, wherein the amino acid is one or more of Lys, Asn, Thr, Ser, He, Met, Pro, His, Gin, Arg, Gly, Asp, Glu, Ala, Vai, Phe, Leu, Tyr, Cys, and/or Trp. In some embodiments, the linker comprises a carbon chain, amide bond or ether bond. In some embodiments, the linker comprises a hydrazone bond, vinyl ether bond, acetal bond, ketal bond or disulphide bond. In certain embodiments, the linker comprises Gly-Phe-Leu-Gly. In some embodiments, the linker comprises Val-Cit (Cit=citrulline). In some embodiments, the linker comprises Phe-Lys. In some embodiments, the linker is polyethylene glycol (PEG) chains, acetate linkers, ester linkers, lectins, buSS (disulfylbutyrate) or maleimide.

[0103] In a further embodiment, provided is a LHRH-PDC comprising the sequence:

X.sub.1-His-Trp-Ser-X.sub.2-X.sub.3(L-D)-X.sub.4-X.sub.5-Pro-NHR [0104] wherein [0105] X.sub.1 is pGlu or Gln; [0106] X.sub.2 is Tyr, Phe or His; [0107] X.sub.3 is D-Lys or D-Lys(Ahx); [0108] X.sub.4 is Leu, Val, Trp or Met; [0109] X.sub.5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; [0110] R is CH.sub.2CH.sub.3 or CH.sub.3; [0111] wherein [0112] L is a linker; and [0113] D is a cytotoxic agent,
or a pharmaceutically acceptable salt thereof. In some embodiments, X.sub.1 is Gln. In some embodiments, X.sub.1 is pGlu. In some embodiments, X.sub.1 is Gln and X.sub.2 is Tyr. In some embodiments, X.sub.1 is Gln and X.sub.2 is Phe. In some embodiments, X.sub.1 is Gln and X.sub.2 is His. In some embodiments, X.sub.3 is D-Lys. In some embodiments, X.sub.3 is D-Lys(Ahx). In some embodiments, X.sub.1 is Gln and X.sub.4 is Leu. In some embodiments, X.sub.1 is Gln and X.sub.4 is Val. In some embodiments, X.sub.1 is Gln and X.sub.4 is Trp. In some embodiments, X.sub.1 is Gln and X.sub.4 is Met. In some embodiments, X.sub.1 is Gln and X.sub.5 is Arg. In some embodiments, X.sub.1 is Gln and X.sub.5 is Gln. In some embodiments, X.sub.1 is Gln and X.sub.5 is Trp. In some embodiments, X.sub.1 is Gln and X.sub.5 is Ser. In some embodiments, X.sub.1 is Gln and X.sub.5 is Leu. In some embodiments, X.sub.1 is Gln and X.sub.5 is Asn. In some embodiments, X.sub.1 is Gln and X.sub.5 is Phe. In some embodiments, X.sub.1 is Gln and X.sub.5 is Tyr. In some embodiments, X.sub.1 is Gln and X.sub.5 is Lys. In certain embodiments, R is CH.sub.2CH.sub.3. In certain embodiments, R is CH.sub.3.

[0114] In certain embodiments, a LHRH peptide derivative may be fused with a cytotoxic agent. In a related embodiment, a LHRH peptide derivatives is fused with a cytotoxic agent via an uncleavable linker.

[0115] In a further embodiment, provided is an antiproliferative LHRH peptide derivative comprising the sequence:

X.sub.1-His-Trp-Ser-X.sub.2-X.sub.3-X.sub.4-X.sub.5-Pro-NHR [0116] wherein [0117] X.sub.1 is pGlu or Gln; [0118] X.sub.2 is Tyr, Phe or His; [0119] X.sub.3 is D-Lys or D-Lys(Ahx); [0120] X.sub.4 is Leu, Val, Trp or Met; [0121] X.sub.5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; and [0122] R is CH.sub.2CH.sub.3 or CH.sub.3,
or a pharmaceutically acceptable salt thereof. In certain embodiments, X.sub.1 is Gln, X.sub.3 is D-Lys and R is CH.sub.2CH.sub.3. In some embodiments, X.sub.1 is pGlu, X.sub.3 is D-Lys and R is CH.sub.2CH.sub.3. In some embodiments, X.sub.1 is pGlu, X.sub.3 is D-Lys(Ahx) and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.1 is Gln, X.sub.2 is Tyr, the X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.1 is pGlu, X.sub.2 is Tyr, X.sub.3 is D-Lys(Ahx), X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3.

[0123] In a further embodiment, provided is a LHRH peptide derivative comprising the sequence:

X.sub.1-His-Trp-Ser-X.sub.2-X.sub.3-X.sub.4-X.sub.5-Pro-NHR [0124] wherein [0125] X.sub.1 is Gln; [0126] X.sub.2 is Tyr, Phe or His; [0127] X.sub.3 is D-Lys or D-Lys(Ahx); [0128] X.sub.4 is Leu, Val, Trp or Met; [0129] X.sub.5 is Arg, Gln, Trp, Ser, Leu, Asn, Phe, Tyr or Lys; and [0130] R is CH.sub.2CH.sub.3 or CH.sub.3,
or a pharmaceutically acceptable salt thereof. In certain embodiments, X.sub.3 is D-Lys and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.2 is Tyr, X.sub.3 is D-Lys, X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, X.sub.2 is Tyr, X.sub.3 is D-Lys(Ahx), X.sub.4 is Leu, X.sub.5 is Arg and R is CH.sub.2CH.sub.3. In certain embodiments, R is CH.sub.2CH.sub.3. In certain embodiments, R is CH.sub.3. In some embodiments, the invention provides a LHRH peptide conjugate comprising a LHRH peptide derivative comprising the sequence: Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 wherein the LHRH peptide derivative is conjugated to a cytotoxic agent via a self immolative linker.

[0131] In some embodiments, the invention provides a LHRH peptide conjugate comprising a LHRH peptide derivative comprising the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 wherein the LHRH peptide derivative is conjugated to a cytotoxic agent via a self immolative linker.

[0132] In some embodiments, the invention provides a LHRH peptide conjugate comprising a LHRH peptide derivative comprising the sequence: pGlu-His-Trp-Ser-Tyr-D-Lys(Ahx)-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 wherein the LHRH peptide derivative is conjugated to a cytotoxic agent via a self immolative linker.

[0133] Further preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings.

EXAMPLES

Example 1: Synthesis of Different LHRH Peptide Derivatives

[0134] The LHRH peptide derivatives of the invention were designed and synthesized on Rink amide resin following the in situ neutralization protocol (P. Varamini et al., J Med Chem., 2017, 60; P. Varamini et al., Int J Pharm, 2017, 521) for Fmoc solid-phase chemistry (the structure of the LHRH peptide derivatives are shown in FIG. 1A). The purity of each of the peptides (LD4, LD5 and LD6) was greater than 98%. The peptides were purified by reverse phase high performance liquid chromatography (RP-HPLC) on a Shimadzu system using a Vydac C18 column (5 mm, 22 250 mm) running a gradient of two solvents, A: H2O, 0.1% TFA, and B: acetonitrile/H2O 9:1, 0.1% TFA. Either a gradient of 20% to 60% B over 60 min (peptides 1-3, 7 and 9-10) or a gradient of 10% to 60% B over 70 min (peptides 4-6 and 8) was used at a flow rate of 10 mL/min. Collected fractions were analyzed by High resolution MS and ESI-MS and analytical RP-HPLC using a Vydac C4 and C18 column (5 mm, 4.6 250 mm) and a gradient of 0% to 100% B over 30 min at a flow rate of 1 mL/min. Pure fractions were combined and lyophilized

Example 2: Synthesis of LD5-mc-vc-PABC-MMAE Conjugate

[0135] The LD5-me-vc-PABC-MMAE conjugate was synthesized by Fmoc solid-phase chemistry techniques (P. Varamini et al., J Med Chem., 2017, 60; P. Varamini et al., Int J Pharm, 2017, 521).

Example 3: Metabolic Stability of LHRH Peptide Derivatives

[0136] The inventors surprisingly found that the LHRH peptide derivatives have improved metabolic stability. The metabolic stability of the native LHRH peptide, LHRH peptide derivatives LD4, LD5 and LD6, and known LHRH agonists triptorelin ([w6]LHRH) and [k6]LHRH were examined in human plasma (FIG. 1B). The LHRH peptide derivatives LD4, LD5 and LD6 exhibit substantially improved half-life (T.sub.1/2 of about 257 min, 365 min and 309 min for LD-4, LD-5 and LD-6, respectively) compared to the native LHRH peptide (T.sub.1/2 of about 10 min) and LHRH agonists that are known in the art, i.e. triptorelin ([w.sup.6] LHRH) and [k.sup.6] LHRH (T.sub.1/2 of about 19 min and 39 min, respectively).

[0137] LD4, LD5 and LD6 show uniquely high stability but among all, LD5 had the highest half-life of 365 min and was selected for conjugation to MMAE via the self-immolative linker, mc-vc-PABC. The binding affinity of the LD5-me-vc-PABC-MMAE conjugate to LHRH receptors was investigated by DuoLink assay. The assay, as shown in FIG. 6, confirmed a high binding affinity of the peptide ligand to LHRH receptors.

Example 4: Anti-Proliferative Activity

[0138] The inventors further discovered that LD4, LD5 and LD6 have significantly higher anti-proliferative activities in 3 different breast cancer cell lines compared to both native LHRH and the agonist [w6]LHRH, which has been used in the clinic for hormone-dependent gynaecological cancers (see Table 1).

Example 5: LHRH Receptor (LHRH-R) is Expressed in Human Breast Cancer Cell Lines

[0139] Cultures from breast cancer cells MDA-MB-231, SK-BR-3 and MCF-7 were harvested and lysed in lysis buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM TrisHCl, pH8.0, and protease inhibitor mixture), and sonicated 15 times for 1 second on ice, followed by centrifugation at 16,100×g at 4° C. for 30 minutes. 50 micrograms of total protein extracts were subjected to 8% SDS/PAGE gel. Following overnight transfer at 4° C., polyvinylidene difluoride (PDVF) membranes were blocked in 5% bovine serum albumin (BSA) for 1 hour, and incubated with anti-LHRH receptor primary antibodies (SolarBio Life Sciences) overnight at 4° C. After washing in Tris buffered saline with Tween (TBS-T), membranes were incubated with horseradish peroxidase-conjugated (HRP-conjugated) secondary antibody for 1 hour. After washing, proteins were detected using ECL-Plus chemiluminescence detection system (GE Healthcare). Density was measured using the Image J program. β-actin was used as control in Western Blot to determine LHRH-R expression in MDA-MB-231, SK-BR-3 and MCF-7 cancer cell lines. Western blot analysis indicated expression of LHRH receptors in 3 breast cancer cell lines with different characteristics (MDA-MB-231: ER−, HER-2−, PR−; MCF-7, ER+, HER-2+, PR+; and SK-BR-3, ER−, HER-2+, PR−). These cell lines were used for the anti-proliferative studies of the LHRH peptide derivatives. This study was performed using the antibody specifically raised against the LHRH-R. A major protein band of approximately 64 kDa molecular mass known for the human pituitary LHRH receptor was identified in MCF-7, MDA-MB-231 and SK-BR-3 (FIG. 2 lanes 1, 2, and 3 respectively). The level of LHRH-R expression relative to 3-actin in MCF-7, MDA-MB-231 and SK-BR-3 showed that these breast cancer cells can be actively targeted through the LHRH receptor.

[0140] Immunocytochemistry was used to confirm the expression of LHRH-Rs in MDA-MB-231 (TNBC cell model), SKOV-3 (low LHRH-R expressing cancer cell model) as well as HMEC and MCF-10A (i.e. normal breast cells) (FIG. 3A). The quantification analysis (FIG. 3B) showed LHRH-R signal intensity is significantly higher in MDA-MB-231 (TNBC cell model) compared to SKOV-3 (low-expressing LHRH-R cancer cell model) and normal breast cells (p<0.05).

TABLE-US-00001 TABLE 1 Direct antiproliferative activity of LHRH peptide derivatives Cell LHRH [w.sup.6]GnRH Line LD4 LD5 LD6 (9) (10) MDA-MB- 36.2 ± 0.8* 38.1 ± 1.4* 33.2 ± 2.5* >100 68.1 ± 8.1 231 (μM) MCF-7 51.3 ± 3.1* 47.6 ± 5.4* 49.2 ± 2.1* >100 79.4 ± 5.3 (μM) SK-BR-3 31.2 ± 4.8* 33.5 ± 2.5* 29.7 ± 1.1* >100 81.2 ± 7.2 IC.sub.50 (μM) SKOV-3 >100 >100 >100 >100 >100 IC.sub.50 (μM)

[0141] The IC.sub.50 values (μM) were estimated from concentration-response curves using non-linear regression for inhibition of cell growth. Data are expressed as mean±SD from at least three independent experiments, each in triplicate. Statistical analysis was performed using a two-way ANOVA (* p<0.05, the IC.sub.50 for each compared with that of their corresponding parent peptide for the same cell line).

Example 6: Cytotoxicity of LD5-mv-vc-PABC-MMAE

[0142] The cytotoxicity of the LHRH peptide derivative pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHCH.sub.2CH.sub.3 (LD5) conjugated with MMAE by the self-immolative linker, mv-vc-PABC (i.e. LD5-mv-vc-PABC-MMAE) was determined with the MMT assay. This assay was used to examine whether LD5-mv-vc-PABC-MMAE affects the proliferation of TNBC cells that expresses LHRH-R. The TNBC cell model, MDA-MB-231, was used to screen for the relative cytotoxicity of LD5-me-vc-PABC-MMAE compared to MMAE and in normal breast cells, HMEC and MCF-10A as well as SKOV-3 (LHRH-R negative controls). Cells were incubated with each compound for 72 h and relative cellular viability was determined using the colorimetric MTT assay. The growth inhibitory effect of LD5-me-vc-PABC-MMAE and MMAE was reported as IC.sub.50 (nM) values for normal breast cells, MDA-MB-231 and SKOV-3 (FIG. 4A).

[0143] Normal breast cells (HMEC and MCF-10A) were sensitive to MMAE with IC.sub.50 value of 0.11 nM and 0.83 nM, respectively. However, LD5-me-vc-PABC-MMAE did not show significant cytotoxic effect on normal breast cells (IC.sub.50 value of >1000 nM). In contrast, the TNBC cell line (MDA-MB-231) showed significant sensitivity to LD5-me-vc-PABC-MMAE with IC.sub.50 value of 1.26 nM. Although the SKOV-3 cell line had higher sensitivity to MMAE with IC.sub.50 value of 0.03 nM, this cell line was resistant to LD5-me-vc-PABC-MMAE with IC.sub.50 value of >1000 nM.

[0144] The cytotoxicity of LD5-mc-vc-PABC-MMAE was assessed in comparison to MMAE for all cells. The potency of LD5-me-vc-PABC-MMAE was significantly lower than MMAE in certain concentrations in normal breast cells (HMEC and MCF-10A), TNBC cells (MDA-MB-231) and LHRH-R negative controls (SKOV-3) (FIGS. 4B, 4C, 4D and 4E). This decrease in cytotoxicity of MMAE when conjugated with LD5 demonstrates the selectivity of the LHRH uptake pathway via the LHRH receptor in comparison to simple diffusion of free MMAE in the cell lines.

Example 7: Role of LHRH Receptor in Cytotoxicity of LD5-mc-vc-PABC-MMAE

[0145] Receptor binding competitive assay was performed to examine the association of cytotoxicity with binding to LHRH-Rs. TNBC cells (MDA-MB-231) were pre-treated with 100 M of triptorelin (TRN) for 2 h to block the LHRH-R. The significant cytotoxic effects of LD5-mc-vc-PABC-MMAE (LM) was reversed after pre-treatment with TRN (FIGS. 5A and 5B). The MTT assay showed that blocking LHRH-R through pre-treatment with TRN significantly increases the cell viability of LD5-me-vc-PABC-MMAE treated cells. This demonstrates the significant role that LHRH-Rs play in LD5-me-vc-PABC-MMAE's anti-cancer activity (FIG. 5).

Example 8: Interaction of LHRH-R and LHRH in LD5-mc-vc-PABC-MMAE Treated Environment

[0146] Proximity ligation assay (PLA) was used to determine the interaction between LHRH-R and LHRH in LD5-mc-vc-PABC-MMAE treated cells. Duolink® Proximity Ligation Assay (PLA) allows in situ detection of endogenous proteins, protein modifications, and protein interactions with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution in unmodified cells and tissues. Typically, two primary antibodies raised in different species are used to detect two unique protein targets. PLA reagents were added to the fixed MDA-MB-231 cells after incubating these cells with the primary antibody specific for LHRH-R and LHRH. In cells treated with LD5-me-vc-PABC-MMAE, the signal localization reveals the protein interaction to be at intracellular sites as well as at the plasma membrane (FIG. 6). This qualitative result confirmed interactions between LHRH-R and its ligand during the LD5-me-vc-PABC-MMAE uptake by MDA-MB-231 cells.

Example 9: In-Vitro Uptake of LD5-mc-vc-PABC-MMAE in TNBC Cells

[0147] The uptake of LD5-me-vc-PABC-MMAE by TNBC cells was examined using the TNBC cell line (MDA-MB-231) which overexpresses LHRH-R. The uptake was compared with normal breast cells (LHRH-R negative control). LD5-me-vc-PABC-MMAE was incubated with MDA-MD-231 normal breast cells for 18 h, and intracellular uptake of the conjugate was monitored using confocal LSM (FIG. 7A).

[0148] As shown in FIG. 7A, there was a significantly higher uptake of LD5-mc-vc-PABC-MMAE in LHRH-R positive cells (MDA-MB-231) compared to normal breast cells (MCF-10A and HMEC). This was supported by quantitative comparisons of the intracellular uptake of LD5-mc-vc-PABC-MMAE in LHRH-R positive and negative cells (FIG. 7B). These data support the active targeted delivery of the compound through LHRH-R in TNBC cells (p<0.05).

Example 10: Effects of LD5-mc-vc-PABC-MMAE on α-Tubulin Polymerisation in Normal and Cancer Cells

[0149] The intracellular effects of MMAE on α-tubulin polymerisation were examined using α-tubulin immunostaining in TNBC cells (MDA-MB-231) and normal breast cells (MCF-10A and HMEC) treated with MMAE and MMAE conjugated with LD5 (LD5-mc-vc-PABC-MMAE). TNBC cells and normal breast cells were fixed after incubating with 1 M of MMAE and LD5-mc-vc-PABC-MMAE for 18 h along with PBS as a control. The α-tubulin was stained by immunostaining and observed by confocal LSM. As shown in FIG. 8A, TNBC cells and normal breast cells treated with MMAE show dramatic decrease in α-tubulin formation compared to PBS control indicating its non-selective activity against normal breast cells and cancer cells (i.e. TNBC cell model). The α-tubulin signal in normal breast cells treated with PBS and LD5-mc-vc-PABC-MMAE (LM) were significantly higher than their MMAE treatment counterpart group (FIG. 8B) indicating the lower impact of the targeted drug on the α-tubulin polymerisation as a factor of cell survival (FIG. 4).

Example 11: Effects of Silencing LHRH-R Gene on the Uptake of LD5-mc-vc-PABC-MMAE by TNBC Cells

[0150] To further examine the role of LHRH-R in the uptake of LD5-me-vc-PABC-MMAE, LHRH-R expression was blocked by silencing their gene in MDA-MB-231 (TNBC cell model). This study was performed by co-transfection of siRNAs (RNAi-Mate transfection reagent and siRNA, GNRHR-homo-2242, GNRHR-homo-2701, and scrambled RNA were from GenePharma) and a fluorescently labelled negative control siRNA (FAM transfection efficiency control was from GenePharma). As shown in FIG. 9, silencing yield over 83% reduction in LHRH-R expression in transfected MDA-MB-231 cells compared to negative control (FIG. 9, p<0.05). The uptake of LD5-me-vc-PABC-MMAE was significantly reduced after silencing the LHRH-R gene expression indicating that the construct is actively targeted to be up-taken by the cancer cells via the overexpressed LHRH-Rs (FIG. 10, p<0.05).

Example 12: In Vitro Metabolic Stability of LD5-(mc-vc-PABC)-MMAE

[0151] The metabolic stability of LD5-(mc-vc-PABC)-MMAE was investigated in cell culture media (c-media), human and mouse plasma. An LC/MS method was developed to detect both free MMAE and the presence of any degraded species from the whole construct, LD5-(mc-vc-PABC)-MMAE. To evaluate the stability of the valine-citrulline linkage, LD5-(mc-vc-PABC)-MMAE at 1 μM was incubated in c-media, human and mouse plasma at 37° C. for a period of 10 days. Aliquots were taken at pre-determined time intervals (t=0, 1, 2, 4, 7, 10 days) and analysed by LC/MS for the release of free MMAE.

[0152] It was shown that LD5-(mc-vc-PABC)-MMAE remained stable during the course of the study, which was 10 days (FIG. 11). This study revealed that less than 3% of the total drug was released as MMAE in human plasma and c-media after 10 days. In mouse plasma, less than 5% of the total drug was released after 10 days. Furthermore, qualitative full-scan LC/MS analysis, including UV detection, of the same samples did not reveal the presence of other molecular species identifiable as drug or drug-linker degradation products.

[0153] The use of a highly stable peptide derivative and an intracellular linker in the design on LHRHD-(mc-vc-PABC)-MMAE (i.e. LD-5-(mc-vc-PABC)-MMAE) resulted in a stable conjugate in human and mouse plasma as well as cell culture media (Example 12). The drug was stably attached to the peptide, showing only around 3% release of MMAE following 10-day incubation in human plasma, but in vitro data showed that it had been cleaved by lysosomal proteases once internalised via LHRH receptors. These stability data were comparable with those of ADCs in the clinic containing MMAE such as Brentuximab vedotin (cAC10-vcMMAE) or Trastuzumab Ermtansine (T-DMI) (J A. Francisco et al., Blood, 2003, 102; B. Bender et al., The AAPS Journal, 2014, 16). While these conjugates with these stability data have been successful in the clinical trials and they are in the market now, there is no report on the in vitro stability of Zoptarelin Doxorubicin (AEZS 108, an LURH receptor-targeted PDC that reached clinical trials). The reason this conjugate failed in phase III clinical trials was lack of stability and release of doxorubicin in the plasma before reaching the tumour site. This led the PDC to have a similar safety profile as that of free doxorubicin. There was no superiority in both toxicity and efficacy over free doxorubicin.

Example 13: In Vivo Minimum Tolerated Dose and Toxicity Studies

[0154] A. Phase I: Single-Dose MTD

[0155] LD5-(mc-vc-PABC)-MMAE was administered IV to groups of 3 female NOD/SCID mice (23±3 g). Animals received an initial dose of 3 mg/kg. If the animals survived for 72 hours, the dose for the next cohort was increased. If one or more animals died, the dose for the next cohort was decreased. The testing stopped when all animals survived at the upper bound, or when two or three dose levels had been tested or when the upper or lower bound had been reached. At each dose level, animals were observed for the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) during the first 15 minutes then again at 1 and 2 hours. Body weights were recorded pre-dose and at 72 hours after treatment. The animals were observed and mortality noted daily after compound administration for 3 days. Gross necropsy was performed on all animals without tissue collection. No significant adverse effects were noted in both 3 and 10 mg/kg through IV injection at all monitored time points (15 minutes, 1 and 2 hours). No mortality and body weight changes were noted, signifying the dose level was tolerated (Tables 2 and 3). The dose at 10 mg/kg was then determined for the following repeat-dose MTD study (Phase II).

TABLE-US-00002 TABLE 2 Maximum Tolerated Dose, Autonomic Signs in Mice (Phase I: Mortality) Dose Mortality (death/test) Compound Route (mg/kg) 15 min 1 hr 2 hr 24 hr 48 hr 72 hr Vehicle IV 5 mL/kg 0/3 0/3 0/3 0/3 0/3 0/3 (PBS) LHRHD-(mc- IV 3 0/3 0/3 0/3 0/3 0/3 0/3 vc-PABC)- 10 0/3 0/3 0/3 0/3 0/3 0/3 MMAE

TABLE-US-00003 TABLE 3 Maximum Tolerated Dose, Autonomic Signs in Mice (Phase I: Body weight) Dose Body Weight (g) Compound Route (mg/kg) No. 0 72 hr Vehicle IV 5 1 22 22 (PBS) mL/kg 2 22 22 3 21 21 LHRHD-(mc- IV  3 1 21 21 vc-PABC)- 2 21 21 MMAE 3 21 21 10 1 20 21 2 20 20 3 20 20

[0156] B. Phase II: Repeat-Dose MTD

[0157] LD-5-(mc-vc-PABC)-MMAE (10 mg/kg; determined by the results by Phase I) was administered IV once weekly on days 1 and 8 to groups of 3 female NOD/SCID mice (23±3 g). Animals were observed for the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) during the first 15 minutes then again at 1 and 2 hours after each treatment on days 1 and 8. Body weights were recorded pre-dose and on days 1, 4, 8, 12 and 15. The animals were observed and mortality noted daily after first compound administration for 15 days. Gross necropsy was performed on all animals without tissue collection. No marked adverse effects were observed at 10 mg/kg IV after the first and the second administrations of LD-5(mc-vc-PABC)-MMAE on Day 1 and 8 (15 minutes, 1 and 2 hours); in addition, all of the tested animals were alive at the termination of the study period, suggesting the dose level were tolerated after the repeat administrations on days 1 and 8 (Tables 3 and 4). No abnormality was found after the gross necropsy in both phases.

TABLE-US-00004 TABLE 4 Maximum Tolerated Dose, Autonomic Signs in Mice - Phase II Mortality Dose Mortality (death/test) Compound Route (mg/kg) Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Vehicle IV 5 mL/kg 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 (PBS) LHRHD-(mc- IV 10 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 vc-PABC)- MMAE Dose Mortality (death/test) Compound Route (mg/kg) Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Vehicle IV 5 mL/kg 0/3 0/3 0/3 0/3 0/3 0/3 0/3 (PBS) LHRHD-(mc- IV 10 0/3 0/3 0/3 0/3 0/3 0/3 0/3 vc-PABC)- MMAE

TABLE-US-00005 TABLE 5 Maximum Tolerated Dose, Autonomic Signs in Mice - Phase II Body Weight Dose Body Weight (g) Compound Route (mg/kg) No. Day 1 Day 4 Day 8 Day 12 Day 15 Vehicle IV 5 mL/kg 1 21 23 24 25 26 (PBS) 2 23 23 23 23 25 3 22 23 24 25 26 LHRHD-(mc- IV 10 1 22 22 22 23 24 vc-PABC)- 2 22 23 23 24 25 MMAE 3 21 23 23 24 25

[0158] The MTD and toxicity studies of LHRHD-(me-v-PABC)-MMAE (i.e. LD-5-(mc-vc-PABC)-MMAE) was performed in female NOD/SCID mice, and no toxicity was observed in any of the mice being dosed at 10 mg/kg. Therefore, the MTD was not reached at this dose. With the corresponding ADCs bearing MMAE the MTD has been considerably lower. For example, MTD for brentuximab vedotin in mice was achieved at 30-40 mg/kg which is equivalent to approximately 70 mg/kg of LHRHD-(mc-vc-PABC)-MMAE. Considering the comparable selectivity and stability of LHRHD-(mc-vc-PABC)-MMAE with the corresponding ADCs, and a markedly lower MTD, a superior safety profile is predicted for this PDC. Other advantages for this PDC over current ADCs are a significantly lower cost of production and simpler manufacturing processes.