ANTI-CTLA4 CONJUGATES

Abstract

The present invention relates to an anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof, wherein said conjugate comprises a plurality of anti-CTLA4 moieties -D covalently conjugated via at least one moiety -L.sup.1-L.sup.2- to a polymeric moiety Z, wherein -L.sup.1- is covalently and reversibly conjugated to -D and -L.sup.2- is covalently conjugated to Z and wherein -L.sup.1- is a linker moiety and -L.sup.2- is a chemical bond or a spacer moiety; and related aspects.

Claims

1. An anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof, wherein said conjugate comprises a plurality of anti-CTLA4 moieties -D covalently conjugated via at least one moiety -L.sup.1-L.sup.2- to a polymeric moiety Z, wherein -L.sup.1- is covalently and reversibly conjugated to -D and -L.sup.2- is covalently conjugated to Z and wherein -L.sup.1- is a linker moiety and -L.sup.2- is a chemical bond or a spacer moiety.

2. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein Z comprises a polymer.

3. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein Z is a hydrogel.

4. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein Z is a PEG-based or hyaluronic acid-based hydrogel.

5. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 3, wherein Z is a hyaluronic acid-based hydrogel.

6. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 3, wherein the hydrogel is non-degradable.

7. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -D is selected from the group consisting of wild-type F, anti-CTLA4 antibodies, Fc enhanced for effector function/FcγR binding anti-CTLA4 antibodies, anti-CTLA4 antibodies conditionally active in tumor microenvironment, anti-CTLA4 small molecules, CTLA4 antagonist fusion proteins, anti-CTLA4 anticalins, anti-CTLA4 nanobodies and anti-CTLA4 multispecific biologics based on antibodies, scFVs or other formats.

8. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -D is ipilimumab.

9. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -D is tremelimumab.

10. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein the anti-CTLA4 conjugate further comprises non-anti-CTLA4 moieties -D.

11. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 10, wherein the non-anti-CTLA4 moieties -D are selected from the group consisting of cytotoxic/chemotherapeutic agents, immune checkpoint inhibitors or antagonists, immune agonists, multi-specific drugs, antibody-drug conjugates (ADC), radionuclides or targeted radionuclide therapeutics, DNA damage repair inhibitors, tumor metabolism inhibitors, pattern recognition receptor agonists, protein kinase inhibitors, chemokine and chemoattractant receptor agonists, chemokine or chemokine receptor antagonists, cytokine receptor agonists, death receptor agonists, CD47 or SIRPα antagonists, oncolytic drugs, signal converter proteins, epigenetic modifiers, tumor peptides or tumor vaccines, heat shock protein (HSP) inhibitors, proteolytic enzymes, ubiquitin and proteasome inhibitors, adhesion molecule antagonists, and hormones including hormone peptides and synthetic hormones.

12. The anti-CTLA4 conjugate of claim 1, wherein -L.sup.1- is of formula (XIII): ##STR00168## wherein the dashed line indicates the attachment to the nitrogen of the primary or secondary amine of -D; v is selected from the group consisting of 0 or 1; —X.sup.1— is selected from the group consisting of —C(R.sup.8)(R.sup.8a)—, —N(R.sup.9)— and —O—; ═X.sup.2 is selected from the group consisting of ═O and ═N(R.sup.10); —X.sup.3 is selected from the group consisting of —O, —S and —Se; each p is independently selected from the group consisting of 0 or 1, provided that at most one p is 0; —R.sup.6, —R.sup.6a, —R.sup.10 are independently selected from the group consisting of —H, —C(R.sup.11)(R.sup.11a)(R.sup.11b) and -T; —R.sup.9 is selected from the group consisting of —C(R.sup.11)(R.sup.11a)(R.sup.11b) and -T; —R.sup.1, —R.sup.1a, —R.sup.2, —R.sup.2a, —R.sup.3, —R.sup.3a, —R.sup.4, —R.sup.4a, —R.sup.5, —R.sup.5a, —R.sup.7, —R.sup.8, —R.sup.5a, —R.sup.11, —R.sup.11a and —R.sup.11b are independently selected from the group consisting of —H, halogen, —CN, —C(O)OR.sup.12, —OR.sup.12, —C(O)R.sup.12, —C(O)N(R.sup.12)(R.sup.12a), —S(O).sub.2N(R.sup.12)(R.sup.12a), —S(O)N(R.sup.12)(R.sup.12a), —S(O).sub.2R.sup.12, —S(O)R.sup.12, —N(R.sup.12)S(O).sub.2N(R.sup.12a)(R.sup.12b), —SR.sup.12, —NO.sub.2, —N(R.sup.12)C(O)OR.sup.12a, —N(R.sup.12)C(O)N(R.sup.12a)(R.sup.12b), —OC(O)N(R.sup.12)(R.sup.12a), -T, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl; wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more —R.sup.13, which are the same or different; and wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.14)—, —S(O).sub.2N(R.sup.14)—, —S(O)N(R.sup.14)—, —S(O).sub.2—, —S(O)—, —N(R.sup.14)S(O).sub.2N(R.sup.14a)—, —S—, —N(R.sup.14)—, —OC(OR.sup.14)(R.sup.14a)—, —N(R.sup.14)C(O)N(R.sup.14a)— and —OC(O)N(R.sup.14)—; —R.sup.12, —R.sup.12a, —R.sup.12b are independently selected from the group consisting of —H, -T, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl; wherein -T, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more —R.sup.13, which are the same or different and wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.14)—, —S(O).sub.2N(R.sup.14)—, —S(O)N(R.sup.14)—, —S(O).sub.2—, —S(O)—, —N(R.sup.14)S(O).sub.2N(R.sup.14a)—, —S—, —N(R.sup.14)—, —OC(OR.sup.14)(R.sup.14a)—, —N(R.sup.14)C(O)N(R.sup.14a)— and —OC(O)N(R.sup.14)—; wherein each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more —R.sup.13, which are the same or different; —R.sup.13 is selected from the group consisting of halogen, —CN, oxo, —C(O)OR.sup.15, —OR.sup.15, —C(O)R.sup.15, —C(O)N(R.sup.15)(R.sup.15a), —S(O).sub.2N(R.sup.15)(R.sup.15a), —S(O)N(R.sup.15)(R.sup.15a), —S(O).sub.2R.sup.1, —S(O)R.sup.15, —N(R.sup.15)S(O).sub.2N(R.sup.15a)(R.sup.15b), —SR.sup.15, —N(R.sup.5)(R.sup.15a), —NO.sub.2, —OC(O)R.sup.15, —N(R.sup.15)C(O)R.sup.15a, —N(R.sup.5)S(O).sub.2R.sup.15a, —N(R.sup.15)S(O)R.sup.15a, —N(R.sup.15)C(O)OR.sup.15a, —N(R.sup.15)C(O)N(R.sup.15a)(R.sup.15b), —OC(O)N(R.sup.15)(R.sup.15a) and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; wherein —R.sup.14, —R.sup.14a, —R.sup.15, —R.sup.15a and —R.sup.15b are independently selected from the group consisting of —H and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; optionally, one or more of the pairs —R.sup.1/—R.sup.1a, —R.sup.2/—R.sup.2a, -R.sup.3/—R.sup.3a, —R.sup.4/—R.sup.4a, —R.sup.5/—R.sup.5a or —R.sup.8/—R.sup.8a are joined together with the atom to which they are attached to form a C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl or an 8- to 11-membered heterobicyclyl; optionally, one or more of the pairs —R.sup.1/—R.sup.2, —R.sup.1/—R.sup.8, —R.sup.1/—R.sup.9, —R.sup.2/—R.sup.9 or —R.sup.2/—R.sup.10 are joined together with the atoms to which they are attached to form a ring -A-; wherein -A- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; optionally, one or more of the pairs —R.sup.3/—R.sup.6, —R.sup.4/—R.sup.6, —R.sup.5/—R.sup.6, —R.sup.6/—R.sup.6a or —R.sup.6/—R.sup.7 form together with the atoms to which they are attached a ring -A′-; wherein -A′- is selected from the group consisting of 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; and each -L.sup.1- is substituted with at least one -L.sup.2- and optionally further substituted provided that the hydrogen marked with the asterisk in formula (XIII) is not replaced by a substituent.

13. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.1- is -L.sup.1- is of formula (XIIIa) ##STR00169## wherein the dashed line indicates attachment to the nitrogen of the primary or secondary amine of -D; —R.sup.1, —R.sup.1a, —R.sup.2, —R.sup.2a, —R.sup.3, —R.sup.3a, —R.sup.5, —R.sup.5a, —R.sup.6 and —R.sup.6a are used as defined in claim 12; and -L.sup.1- is substituted with at least one moiety -L.sup.2- and is optionally further substituted, provided that the hydrogen marked with the asterisk in formula (XIIIa) is not replaced by a substituent.

14. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.1- is of formula (XIIIb) ##STR00170## wherein the dashed line indicates attachment to the nitrogen of the primary or secondary amine of -D; and -L.sup.1- is substituted with at least one moiety -L.sup.2- and is optionally further substituted, provided that the hydrogen marked with the asterisk in formula (XIIIb) is not replaced by a substituent.

15. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.1- is of formula (XIIIc) ##STR00171## wherein the unmarked dashed line indicates attachment to the nitrogen of the primary or secondary amine of -D, and the dashed line marked with # indicates attachment to -L.sup.2-.

16. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.2- is a spacer moiety.

17. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.2- is a spacer moiety selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y1)—, —S(O).sub.2N(R.sup.y1)—, —S(O)N(R.sup.y1)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y1)S(O).sub.2N(R.sup.y1a)—, —S—, —N(R.sup.y1)—, —OC(OR.sup.y1)(R.sup.y1a)—, —N(R.sup.y1)C(O)N(R.sup.y1a)—, —OC(O)N(R.sup.y1)—, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T-, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y3)—, —S(O).sub.2N(R.sup.y3)—, —S(O)N(R.sup.y3)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y3)S(O).sub.2N(R.sup.y3a)—, —S—, —N(R.sup.y3)—, —OC(OR.sup.3)(R.sup.y3a)—, —N(R.sup.y3)C(O)N(R.sup.y3a)—, and —OC(O)N(R.sup.y3)—; —R.sup.y1 and —R.sup.y1a are independently of each other selected from the group consisting of —H, -T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different, and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y4)—, —S(O).sub.2N(R.sup.y4)—, —S(O)N(R.sup.y4)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y4)S(O).sub.2N(R.sup.y4a)—, —S—, —N(R.sup.y4)—OC(OR.sup.4)(R.sup.y4a)—, —N(R.sup.y4)C(O)N(R.sup.y4a)—, and —OC(O)N(R.sup.y4)—; each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more —R.sup.y2, which are the same or different; each —R.sup.y2 is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.y5, —OR.sup.y5, —C(O)R.sup.y5, —C(O)N(R.sup.y5R.sup.y5a), —S(O).sub.2N(R.sup.y5R.sup.y5a), —S(O)N(R.sup.y5R.sup.y5a), —S(O).sub.2R.sup.y5, —S(O)R.sup.y5, —N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), —SR.sup.y5, —N(R.sup.y5R.sup.y5a), —NO.sub.2, —OC(O)R.sup.5, —N(R.sup.y5)C(O)R.sup.y5a, —N(R.sup.y5)S(O).sub.2R.sup.y5a, —N(R.sup.y5)S(O)R.sup.y5a, —N(R.sup.y5)C(O)OR.sup.y5a, —N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b), —OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each —R.sup.y3, —R.sup.y3a —R.sup.y4, —R.sup.y4a, —R.sup.y5, —R.sup.y5a and —R.sup.y5b is independently selected from the group consisting of —H, and C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

18. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein L.sup.2- comprises a moiety ##STR00172##

19. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.2- has a chain length of 1 to 20 atoms.

20. The anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sup.2- comprises a moiety of formula (XIV) ##STR00173## wherein the dashed line marked with the asterisk indicates attachment to -L.sup.1- and the unmarked dashed line indicates attachment to the remainder of -L.sup.2- or to Z.

21. A pharmaceutical composition comprising the anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1.

22-26. (canceled)

27. A method of treating in a mammalian patient in need of the treatment of one or more diseases which can be treated with an anti-CTLA4 drug, comprising the step of administering to said patient in need thereof a therapeutically effective amount of the anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof of claim 1.

28. The method of claim 27, wherein the disease which can be treated with an anti-CTLA4 drug is a cell proliferation disorder.

29. The method of claim 28, wherein the cell proliferation disorder is cancer.

30. The method of claim 27, wherein the anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof is administered together with one or more further drug molecules or treatments.

31. The method of claim 30, wherein the one or more further drug molecules or treatments are administered to said patient prior to, together with or after administration of the anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof.

Description

EXAMPLE 1: SYNTHESIS OF CARBAMATE 2

[1384] ##STR00152##

[1385] 4-Hydroxybenzyl alcohol (1.70 g; 13.69 mmol; 1.00 eq.) was dissolved in THF (20.5 mL) and DIPEA (4.8 mL; 27.39 mmol; 2.00 eq.) was added with stirring. 4-Nitrophenyl chloroformate (2.90 g; 14.38 mmol; 1.05 eq.) in THF (5 mL) and was added dropwise over 25 min. The reaction was stirred for additional 20 minutes at room temperature. N,N,N′-trimethylethylenediamine (2.21 mL; 17.12 mmol; 1.25 eq.) was slowly added to the solution and the reaction mixture was stirred for additional 30 min. The reaction was cooled in an ice-bath, quenched with TFA (3.17 mL; 41.08 mmol; 3.00 eq.) and diluted with water. The aqueous phase was washed with ethyl acetate (3×100 mL). The aqueous phase was lyophilized to yield an oily residue. The residue was co-evaporated with ethyl acetate (3×), dissolved in DCM and dried (Na.sub.2SO.sub.4). After filtration the solvent was evaporated and the oily residue was dried under high vacuum (2 h). A QC by LC-MS revealed a purity of 2 of 94% at 215 nm. The crude material was used in the next step without purification. 11.76 g crude TFA salt of carbamate 2 (max. 13.69 mmol, max. purity of 43 wt %) were obtained.

EXAMPLE 2: SYNTHESIS OF PFP-CARBONATE 3

[1386] ##STR00153##

[1387] Carbamate 2 (11.76 g; 13.69 mmol; 1.00 eq.) was dissolved in acetonitrile (24 mL) and the solution was cooled in an ice-bath. Bis(pentafluorophenyl) carbonate (10.15 g; 25.75 mmol; 1.88 eq.), DMAP (315 mg; 2.58 mmol; 0.19 eq.) and DIPEA (9.0 mL; 51.53 mmol; 3.76 eq.) were added with stirring. The reaction mixture was stirred for 15 minutes. Formation of product 3 was confirmed by LC-MS. The reaction mixture was cooled to −15° C. and was quenched with a mixture of water with 0.1% TFA (12.4 mL) and neat TFA (3.9 mL; 51.48 mmol; 3.76 eq.). The yellow solution was purified by RP-LPLC. The pure fractions were combined, frozen and lyophilized to yield 4.73 g TFA salt of PFP-carbonate 3 as yellow oil (8.21 mmol, 60% over 3 steps).

EXAMPLE 3: PREPARATION OF FMOC PROTECTED AMINE 6

[1388] ##STR00154##

[1389] Fmoc-N-Me-Asp(tBu)-OH (4, 6.96 g; 16.36 mmol; 1.00 eq.) was dissolved in DMF (139 mL). PyBOP (12.77 g; 24.54 mmol; 1.50 eq.) and DIPEA (14.3 mL; 81.79 mmol; 5.00 eq.) were added. Finally, N-Boc-N-methyl-1,3-diaminopropane hydrochlorid (5, 4.04 g; 17.99 mmol; 1.10 eq.) was added and the reaction mixture was stirred at room temperature for 1 hour. Complete conversion to the product was observed by LCMS. The reaction mixture was diluted with 385 mL of dichloromethane and was washed three times with 385 mL of 0.1 N HCl. The organic layer was washed two times with 385 mL of saturated NaHCO.sub.3 solution and once with 200 mL of brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated. The residue was dried under high vacuum overnight to yield the crude product as orange oil (16.25 g). The product was purified by normal phase flash chromatography. The product containing fractions were pooled and the solvent was evaporated. The final material was dried under high vacuum overnight to yield amide 6 (8.59 g, 14.42 mmol, 88%) as white foam.

EXAMPLE 4: PREPARATION OF AMINE 7

[1390] ##STR00155##

[1391] Fmoc protected amine 6 (8.59 g; 14.42 mmol; 1.00 eq.) was dissolved in THF (125 mL). DBU (2.50 mL; 16.73 mmol; 1.16 eq.) was added and the mixture was stirred at room temperature for 12 minutes. An LC-MS chromatogram showed complete conversion of the starting material. The solvent was evaporated. The residue was dissolved in 15 mL of ethyl acetate and purified by flash chromatography. The product containing fractions were pooled and the solvent was evaporated. The final material was dried under high vacuum for 1 hour to yield amine 7 (5.07 g, 13.57 mmol, 94%) as colorless oil.

EXAMPLE 5: COUPLING OF FMOC-ADO-OH TO AMINE 7

[1392] ##STR00156##

[1393] Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH) (5.76 g; 14.93 mmol; 1.10 eq.) and PyBOP (7.77 g; 14.93 mmol; 1.10 eq.) were dissolved in 38 mL of dichloromethane. Then DIPEA (7.09 mL; 40.72 mmol; 3.00 eq.) was added and the carboxylic acid was activated for 1 minute. A solution of amine 7 (5.07 g; 13.57 mmol; 1.00 eq.) in 38 mL of dichloromethane was added to the activated carboxylic acid and the reaction mixture was stirred at room temperature for 2 h. LC-MS analysis showed complete conversion of the starting material. The reaction mixture was diluted with 785 mL of ethyl acetate and was washed three times with 630 mL of 0.1 N HCl. The organic layer was washed once with 471 mL of brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated. The residue was dried under high vacuum for three days (13.59 g crude material). The residue was dissolved in 20 mL of ethyl acetate and purified by flash chromatography. The product containing fractions were pooled and the solvent was evaporated. The final material was dried under high vacuum overnight to yield amide 9 (8.59 g, 11.59 mmol, 85%) as white foam.

EXAMPLE 6: SYNTHESIS OF LINKER CORE UNIT 9

[1394] ##STR00157##

[1395] Reagent 8 (2.19 g; 2.96 mmol; 1.00 eq.) was dissolved in dichloromethane (26 mL). DBU (512 μL; 3.43 mmol; 1.16 eq.) was added to the solution and stirred for 10 min at room temperature. A solution of 3-Maleimidopropionic acid N-hydroxysuccinimide ester (1.18 g; 4.43 mmol; 1.50 eq.) in dichloromethane (46 mL) was added to the reaction mixture. The solution was stirred for 1 min. The reaction mixture was diluted with 500 mL of ethyl acetate. The organic phase was washed twice with a mixture of 400 mL of 0.5% citric acid solution and 100 mL of brine. The organic layer was dried over MgSO.sub.4, filtered and the solvent was evaporated (3.16 g crude material). The crude material was dissolved in 10 mL of ethyl acetate and purified by flash chromatography. Product containing fractions were pooled and the solvent was evaporated to yield linker core 9 (1.68 g, 2.51 mmol, 85%) as oil.

EXAMPLE 7: DEPROTECTION OF LINKER CORE UNIT 9

[1396] ##STR00158##

[1397] Linker 9 (2.84 g; 4.24 mmol; 1.00 eq.) was dissolved in dichloromethane (28.4 mL) and TFA (28.4 mL) was added. The reaction mixture was stirred for 70 minutes at room temperature. Volatiles were removed in a stream of argon and the resulting residue was dried under controlled conditions (rotary evaporator at 40° C. and 12 mbar for 20 min, then high vacuum at room temperature for 45 min). Crude intermediate 10 was immediately used in the next step without further purification. Crude yield was determined as 5.43 g (maximal 4.24 mmol, thus maximal 49% purity of the TFA salt of 10)

EXAMPLE 8: COUPLING OF PFP-CARBONATE 3 TO LINKER 10

[1398] ##STR00159##

[1399] PFP-carbonate 3 (3.18 g; 5.51 mmol; 1.30 eq.) was dissolved in acetonitrile (28.4 mL), the solution was cooled in an ice bath and DIPEA (7.4 mL; 42.40 mmol; 10.00 eq.) was added. A solution of crude intermediate 10 (5.43 g crude, 4.24 mmol; 1.00 eq) in acetonitrile (28.4 mL) was added dropwise over 10 min to the stirred reaction mixture. After 5 min stirring in the ice bath, TFA (1.6 mL; 21.20 mmol; 5.00 eq.) was added to quench the reaction. The reaction mixture was concentrated and the residue dissolved in 1:1 MeCN/water+0.1% TFA (6 mL) and water+0.1% TFA (6 mL). The crude mixture was purified by RP-LPLC. Product containing fractions were combined, frozen and lyophilized to yield 3.16 g TFA salt of linker 11 (3.49 mmol, 82%).

EXAMPLE 9: NHS ACTIVATION OF LINKER 11

[1400] ##STR00160##

[1401] Linker 11 (3.08 g; 3.40 mmol; 1.00 eq.) was dissolved in dichloromethane (31 mL). N-Hydroxysuccinimide (1.18 g; 10.24 mmol; 3.01 eq.), EDC*HCl (1.96 g; 10.2 mmol; 3.00 eq.) and DMAP (41 mg; 0.34 mmol; 0.10 eq.) were added and the reaction mixture was stirred for 1 h at room temperature. The reaction mixture was diluted with 90 mL of dichloromethane and was washed with 90 mL of acidic brine (250 mL brine were acidified with 2.5 mL 1 M HCl, this solution was saturated with additional NaCl). The aqueous phase was extracted with 60 mL of dichloromethane (pH-value aqueous phase 3-3.5). The combined organic phases were dried over Na.sub.2SO.sub.4 and filtered. TFA (0.26 mL; 3.40 mmol; 1.00 eq.) was added. The solvent was removed (rotary evaporator, 40° C., approx. 20 min) to yield 3.53 g of raw product as white foam. The crude product was dissolved in 8 mL of anhydrous acetonitrile (total volume approx. 10 mL, yellowish solution) and purified by LPLC. Product containing fractions were immediately cooled (ice bath) and pooled. The product containing fractions were frozen and lyophilized as soon as possible. The lyophilized, dry material was combined with anhydrous dichloromethane (circa 81 mL in total). The solvent was carefully removed (rotary evaporator, 40° C., foam formation) and dried under high vacuum for 30 min to yield linker 12 as colorless foam with a yield of 2.86 g (2.85 mmol, 84%), 78% purity at 215 nm.

[1402] The product was stored under argon at −80° C. for 16 h. The material was brought to room temperature and dissolved in 28.5 mL of anhydrous DMSO to yield a “100 mM” solution. (No volume correction for the dissolved material was applied). The DMSO solution was sterile filtered (PTFE syringe filters, Millipore Millex-LG, 25 mm, 0.2 μm) to yield about 30 mL of a clear, colorless solution. The material was stored in aliquots under argon at −80° C.

EXAMPLE 10: SYNTHESIS OF BACKBONE REAGENT 13

[1403] Backbone reagent 13 was synthesized as HCl salt using L-lysine building blocks, analogously to an earlier described procedure (WO2013/053856, example 1, compound 1 g therein):

##STR00161##

EXAMPLE 11: SYNTHESIS OF CROSS-LINKER REAGENT 14C

[1404] Cross-linker reagent 14c was synthesized as shown below. Theoretical calculations of the Mw of the polydisperse PEG conjugates were exemplarily performed for a PEG 3300 with an assumed average Mw of 3300 g/mol. For LC-MS analyses, exact masses of the most abundant PEG molecule species with n=77 or 78 ethylene glycol units, were used.

##STR00162##

[1405] Azelaic acid monobenzyl ester (11.8 g, 42.4 mmol, 3.5 eq.) and PEG3300 (40.0 g, 12.1 mmol, 1.0 eq.) were dissolved in DCM (64 mL) and cooled to 0° C. Under stirring, a solution of DCC (8.75 g, 42.4 mmol, 3.5 eq.) and DMAP (74 mg, 0.61 mmol, 0.05 eq.) in DCM (32 mL) was added and the reaction mixture was stirred at room temperature for 17 hours. The mixture was cooled to 0° C. and the precipitated DCU was removed by filtration. The solvent was evaporated in vacuo completely and the residue was dissolved in DCM (50 mL). MTBE (450 mL) was added and the mixture was cooled to −30° C. The precipitate was collected by filtration, washed with pre-cooled MTBE (−20° C., 500 mL) and dried in high vacuum to yield intermediate 14a (41.8 g, 10.9 mmol, 90%).

[1406] MS: m/z 795.88=[M+5H].sup.5+, (calculated monoisotopic mass: [M]=3972.34, n=78)

[1407] Palladium on charcoal (10% Pd, 199 mg) was added to a solution of intermediate 14a (41.6 g, 10.9 mmol) in EtOAc (280 mL). Under stirring, hydrogen was passed through the mixture for 3 minutes. The mixture was then stirred under hydrogen atmosphere for 16 hours. After removal of the catalyst by filtration through a pad of Celite® 503, all volatiles were removed from the filtrate in vacuo to give intermediate 14b (36.8 g, 10.1 mmol, 93%).

[1408] MS: m/z 751.05=[M+5H].sup.5+, (calculated monoisotopic mass: [M]=3748.22, n=77)

[1409] Within one minute and under stirring, DIPEA (5.2 g, 40.4 mmol, 4.0 eq.) was added dropwise to a slightly turbid solution of intermediate 14b (36.8 g, 10.1 mmol, 1.0 eq.) and TSTU (12.2 g, 40.4 mmol, 4.0 eq.) in DCM (110 mL). After one hour, the reaction mixture was filtered through a PE frit in a syringe and the filtrate diluted with DCM (110 mL). The organic phase was washed with a solution prepared from NaOH (3 g) and NaCl (197 g) in water (750 g). Afterwards, the organic phase was dried over MgSO.sub.4, filtered and freed from all volatiles in vacuo. The crude product was dissolved in toluene (260 mL), whereupon an orange-colored solid precipitated, which was removed by filtration. MTBE (500 mL) was added to the filtrate and the mixture was cooled to −20° C. overnight. The precipitate was collected by filtration and dried in high vacuum for three days to yield crosslinker 14c (34.9 g, 9.1 mmol, 90%).

[1410] MS: m/z 798.66=[M+5H].sup.5+, (calculated monoisotopic mass: [M]=3986.28, n=78)

EXAMPLE 12: SYNTHESIS OF PEG-HYDROGEL MICROPARTICLES 15A, 15B AND 15C CONTAINING FREE AMINO GROUPS

[1411] A cylindrical 250 mL reactor with bottom outlet, diameter 60 mm, equipped with baffles, was charged with an emulsion of Cithrol™ DPHS (266 mg) in heptane (80 mL). The reactor content was stirred with a pitch-blade stirrer, diameter 45 mm, at 420 rpm, at room temperature. A solution of cross-linker 14c (2373 mg) and backbone reagent 13 (550 mg) in DMSO (26.39 g) was added to the reactor and stirred for 10 min to form an emulsion. TMEDA (2.5 mL) was added to effect polymerization and the mixture was stirred at room temperature for 40 h. Acetic acid (3.8 mL) was added while stirring. After 10 min, a sodium chloride solution (15 wt %, 100 mL) was added under stirring. After 10 min, the stirrer was stopped and phases were allowed to separate. After 30 min, the aqueous phase containing the PEG-hydrogel microparticles was drained.

[1412] For microparticle classification, the water-hydrogel suspension was diluted with ethanol (40 mL) and wet-sieved on 100, 75, 63, 50 and 40 μm (mesh opening) stainless steel sieves, diameter 200 mm using a sieving machine for 15 min. Sieving amplitude was 1.5 mm, liquid flow was 250 mL/min. Water (4000 mL) was used as the liquid for wet-sieving. The bead fractions on the different sieves were transferred into 50 mL Falcon tubes (max. 14 mL bead suspension per tube) and successively washed with AcOH (0.1% v/v, 3× approx. 40 mL) and ethanol (8× approx. 40 mL) by addition, shaking, centrifugation and decantation. The bead fractions from the sieves with 50, 63 and 75 μm mesh openings were transferred into 20 mL syringes with PE frits and dried in high vacuum for three days to yield amine hydrogels 15a, 15b and 15c. The amine content of the hydrogels was determined for bead fraction 15a, representatively for all batches, by conjugation of an Fmoc-amino acid to the free amino groups on the hydrogel and subsequent Fmoc determination. The following yields were obtained: 15a (50 μm sieve fraction): 183 mg; 15b (63 μm sieve fraction): 398 mg; 15c (75 μm sieve fraction): 337 mg. Amine content was determined as 0.210 mmol/g.

EXAMPLE 13: SYNTHESIS OF MTS-PEG12-NHS ESTER 16C

[1413] ##STR00163##

[1414] 6-Bromohexanoic acid (5.89 g, 30.2 mmol, 1.0 eq.) and sodium methanethio-sulfonate (4.05 g, 30.2 mmol, 1.0 eq.) were dissolved in anhydrous DMF (47.1 mL) under argon atmosphere and stirred at 80° C. for three hours. After cooling to r.t., the mixture was diluted with water (116 mL) and extracted with diethyl ether (3×233 mL). The combined organic layers were washed with brine (350 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure to a volume of 40 mL. The solution was split and added to two portions of cold n-heptane (2×1150 mL) and the mixtures were cooled to −18° C. overnight. The supernatant solutions were decanted and the precipitates were dissolved in diethylether (80 mL combined). This solution was split and added to two portions of cold n-heptane (2×1000 mL) and the mixtures were cooled to −18° C. for two hours. The precipitate was collected by filtration and dried in high vacuum overnight to yield intermediate 16a (5.62 g, 24.8 mmol, 82%).

[1415] MS: m/z 249.02=[M+Na].sup.+, (calculated monoisotopic mass: [M]=226.03)

[1416] DIPEA (2.76 mL, 15.9 mmol, 3.28 eq.) was added to a stirring solution of 16a (1.15 g, 5.08 mmol, 1.05 eq.) and PyBOP (2.64 g, 5.08 mmol, 1.05 eq.) in anhydrous DCM (54.8 mL). After stirring for 30 minutes, 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid (2.99 g, 4.84 mmol, 1.00 eq.) was added and the mixture was stirred at room temperature for additional 30 minutes. Cold MTBE (55 mL) was added to the slightly yellow reaction mixture and it was cooled to −20° C. overnight. No precipitate was formed. All volatiles were removed in vacuo and the residue was dissolved in DCM. After addition of TFA (1.2 mL), the solution was concentrated to 10 mL. Cold MTBE (55 mL) was added to the slightly yellow solution and it was cooled to −20° C. overnight. The supernatant was decanted and the yellow precipitate was washed with cold MTBE (55 mL). The now white residue was dried on the rotavapor. After further purification by preparative RP-HPLC, intermediate 16b (2.81 g, 3.40 mmol, 70%) was obtained as white solid.

[1417] MS: m/z 826.35=[M+H].sup.+, (calculated monoisotopic mass: [M]=825.39)

[1418] 16b (2.81 g, 3.40 mmol, 1.0 eq.), HOSu (470 mg, 4.08 mmol, 1.2 eq.), DMAP (41.6 mg, 0.34 mmol; 0.1 eq.) and DCC (842 mg, 4.08 mmol, 1.2 eq.) were dissolved in anhydrous DCM (32.6 mL) and the mixture was stirred at room temperature for 30 minutes. The precipitated DCU was removed by filtration and the solvent was evaporated from the filtrate. The residue was purified by preparative RP-HPLC to yield pure handle reagent 16c (1.74 g; 1.88 mmol, 55%).

[1419] MS: m/z 923.45=[M+H].sup.+, (calculated monoisotopic mass: [M]=922.40)

EXAMPLE 14: SYNTHESIS OF MTS-FUNCTIONALIZED HYDROGEL 17

[1420] A PEG-hydrogel, comparable to 15c (500 mg, amine content: 0.212 mmol/g, 0.106 mmol, 1.0 eq.), present as a suspension in a mixture of NMP/n-propylamine (99:1 v/v) was partitioned between five 20 mL syringe reactors with PE frits in equal aliquots. Each hydrogel portion was successively washed with anhydrous NMP (5×8 mL), NMP/DIPEA (99:1 v/v, 5×8 mL) and all solvents were expelled completely after complete washing. To each hydrogel portion, an aliquot of 2.46 mL of a freshly prepared solution of 16c (295 mg, 0.32 mmol, 3.0 eq.) in anhydrous NMP (12 mL) and NMP/DIPEA (99:1 v/v, 500 μL) were drawn. The syringe reactors were agitated at 500 rpm for 180 minutes. The reaction mixtures were expelled from all syringes and each hydrogel portion was successively washed with anhydrous NMP (5×8 mL), water containing 0.1% AcOH and 0.01% Tween 20 (5×8 mL) and 20 mM succinate 0.01% Tween 20 pH 4.0 buffer (5×8 mL). The hydrogel aliquots were combined in a 50 mL Falcon tube with additional 20 mM succinate 0.01% Tween 20 pH 4.0 buffer. After brief centrifugation, the volume of the suspension was adjusted to 25 mL by removing an adequate volume of the clear supernatant to yield a suspension of MTS-hydrogel 17 in 20 mM succinate 0.01% Tween 20 pH 4.0 buffer with 25 mL volume and a hydrogel content of 23.0 mg/mL. The MTS load for dry hydrogel was determined as 0.161 mmol/g.

EXAMPLE 15: PREPARATION OF HHC.SUP.MET.-LINKER CONJUGATE MIXTURE 18

[1421] ##STR00164##

[1422] 35 mL of HHC.sup.MET (depicted above as HHC.sup.MET-NH.sub.2) at 4.5 mg/mL in PBS buffer was used in this example. HHC.sup.MET was concentrated, and protein concentration was determined. 14.47 mL HHC.sup.MET in PBS, pH 7.4 at a concentration of 9.7 mg/mL were prepared. 38 mol eq. (2.64 mL) of linker reagent 12 (example 9) (corrected with respect to NHS content, nominal 100 mM stock solution in DMSO) relative to the amount of HHC.sup.MET were added in 30 seconds intervals (4×0.66 mL) to 14.38 mL of the HHC.sup.MET solution. The reaction mixture was mixed carefully after each addition of linker reagent 12 and incubated in total for 8 min at ambient temperature counting from the first addition. The reaction yielded a mixture of unmodified HHC.sup.MET and protected HHC.sup.MET-linker conjugates (e.g. monoconjugates, bisconjugates) 18 (only monoconjugate is exemplary shown above).

[1423] The linker-conjugation reaction was immediately followed by a pH shift towards about pH 4 and a buffer exchange was performed to remove excess linker species from the HHC.sup.MET/HHC.sup.MET-linker conjugate mixture 18. The buffer shift was achieved by addition of 0.047 vol. eq. (0.676 mL) of 0.4 M succinic acid pH 3.0 with respect to the volume of the HHC.sup.MET solution (14.38 mL), and the solution was mixed carefully end-over-end. The buffer exchange to 20 mM succinic acid, pH 4.0 was performed using an Äkta purifier 100 system equipped with a GE HiPrep column at a flow rate of 8.0 mL/min. Four runs with approx. 4.5 mL injection volume per run were performed. After buffer exchange, the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 18 was concentrated using VivaSpin Turbo 15, MWCO 5 kDa centrifugal filters yielding a solution of 11.9 g with a concentration of 10.96 mg/mL.

[1424] To estimate the content of protected HHC.sup.MET-linker conjugates within the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 18, an HPLC-ESI-MS analysis was performed. 0.27 μL of the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 18 (c=10.96 mg/mL) were injected into Waters Acquity UPLC coupled to a Thermo LTQ Orbitrap Discovery high resolution/high accuracy mass spectrometer equipped with TOSOH TSKgel SuperAW3000 column (flow rate 0.4 mL/min, solvent A: UP-H.sub.2O+0.05% TFA, solvent B: UP-Acetonitrile +0.04% TFA, isocratic elution with 50% solvent A at 60° C.).

[1425] The comparison of the relative intensities of the MS peaks corresponding to the unmodified HHC.sup.MET (calculated m/z 1648.76 for [M+16H].sup.16+ ion), mono- (calculated m/z 1697.09 for [M+16H].sup.16+ ion) and bis-conjugate (calculated m/z 1745.43 for [M+16H].sup.16+ ion) provided an estimate of the presence of about 45% of HHC.sup.MET-linker monoconjugates within the HHC.sup.MET/HHC.sup.MET-linker conjugate mixture 18.

[1426] After analysis, 11.9 mL of the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 18 (c=10.96 mg/mL) were pH adjusted to pH 5.5 for hydrogel loading by addition of 0.154 vol. eq. 0.5 M succinic acid, pH 6.2 (1.83 mL). To reach the desired content of EDTA (target concentration 5 mM) and Tween20 (target concentration 0.01%) the obtained solution was supplemented with 1/19 vol. eq. 20 mM succinic acid, 100 mM EDTA, 0.2% Tween20, pH 5.5 (0.722 mL) and the solution was mixed end-over-end. The final volume after pH and buffer adjustment was 14.51 mL with a theoretical concentration of 9.05 mg/mL.

EXAMPLE 16: SYNTHESIS OF TRANSIENT HHC.SUP.MET.-LINKER-HYDROGEL PRODRUG 20

[1427] ##STR00165##

[1428] Conjugation of HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 18 to the reduced thiol functionalized PEG hydrogel 19 was performed by addition of HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture to aim for 65% (w/w) protein content within HHC.sup.MET-linker-hydrogel conjugate.

[1429] 1.33 mL of MTS functionalized PEG hydrogel 17 (23.8 mg/mL nominal gel content with a thiol content of 0.161 mmol/g) in 20 mM succinic acid, 0.01% Tween20, pH 4.0 were transferred into a 20 mL syringe with a frit. The thiol functionalized PEG hydrogel beads were reduced by replacement of the storage solution by 10 mL of 50 mM TCEP solution in PBS-T and incubation for 15 minutes at ambient temperature. Afterwards, the 50 mM TCEP solution was removed from the syringe, and thiol functionalized hydrogel beads were washed in the syringe 10 times with 5 mL 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5 to yield 19. Afterwards, 14.51 mL of the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 18 (c.sub.theoretical=9.05 mg/mL, 131.3 mg) at pH 5.5 were drawn up into the syringe. The resulting suspension was mixed well and incubated at ambient temperature under gentle rotation overnight yielding protected transient HHC.sup.MET-linker hydrogel prodrug 20.

[1430] After overnight incubation, the protected transient HHC.sup.MET-linker hydrogel prodrug 20 was washed in the syringe once with 5 mL 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5 and two times with 5 mL 10 mM iodoacetamide in 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4. The remaining free thiol groups in the prodrug 20 were blocked by 60 minutes incubation with gentle rotation in 30 mM sodium phosphate, 10 mM iodoacetamide, 50 mM TriMED, 0.01% Tween20, pH 7.4 buffer in the syringe at ambient temperature. Removal of iodoacetamide blocking solution was accomplished via ten washing steps in the syringe with 5 mL 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4.

[1431] For deprotection of the protected transient HHC.sup.MET-linker hydrogel prodrug, 5 mL 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4 buffer were drawn up into the syringe and the resulting suspension was incubated at 25° C. overnight in the syringe yielding transient HHC.sup.MET-linker hydrogel prodrug 21.

[1432] Final formulation of transient HHC.sup.MET-linker hydrogel prodrug 21 was achieved by washing the transient HHC.sup.MET-linker hydrogel prodrug 21 ten times in the syringe with 5 mL 20 mM succinic acid, 0.01% Tween20, pH 4.0. The suspension containing transient HHC.sup.MET-linker hydrogel prodrug 21 was transferred to a 5 mL Eppendorf tube and a dense suspension of the transient HHC.sup.MET-linker hydrogel prodrug 21 in 20 mM succinic acid, 0.01% Tween20, pH 4.0 was prepared by removal of the supernatant.

EXAMPLE 17: SYNTHESIS OF PROTECTED DIAMINO ALCOHOL 22B

[1433] ##STR00166##

[1434] 3 (352 mg, 0.61 mmol) was dissolved in acetonitrile (2.50 mL) and the solution cooled in an ice-bath. DIPEA (242 μL, 1.39 mmol) was added and the reaction was mixed. 22a 1,3-diamino-2-propanol (25 mg, 0.28 mmol) was dissolved in acetonitrile (1.00 mL) and added to the reaction. The reaction was mixed and incubated in the ice-bath. A reaction control after 5 min indicated complete reaction.

[1435] After ca. 15 min TFA (106 μL, 1.39 mmol) was added to the ice cooled reaction. The reaction was diluted with 4 mL water containing 0.1% TFA. The product 22b was purified by RP-HPLC.

[1436] Yield: 204 mg (84%, 2× TFA salt)

[1437] MS: m/z 647.34=[M+H].sup.+, (calculated=647.34).

EXAMPLE 18: SYNTHESIS OF LINKER REAGENT 23G

[1438] ##STR00167##

[1439] 23a (1.5 g, 5.7 mmol) was dissolved in THF (37.5 mL). TSTU (2.6 g 8.6 mmol) and DIPEA (3.97 mL, 22.8 mmol) were added. Upon stirring a turbid suspension was formed. The mixture was stirred for 22 h. TSTU (1.7 g 5.5 mmol), DIPEA (2 mL, 11.5 mmol) and DMF (13 mL) were added and the color of the reaction turned dark brown. After a total of 26 h the reaction mixture was diluted with 350 mL of ethyl acetate and washed with 2×200 mL 0.1N HCl and 1× with 100 mL of brine. The organic phase was dried over Na.sub.2SO.sub.4 and evaporated.

[1440] The residue was dried under high vacuum overnight. The product was purified using flash chromatography yielding 23b as colorless oil.

[1441] Yield: 1.65 g (81%)

[1442] MS: m/z 361.17=[M+H].sup.+, (calculated=361.16).

[1443] 23b (1.65 g, 4.58 mmol) was dissolved in DCM (11.6 mL) and N-Me-L-Asp(tBu)-OH (932 mg, 4.59 mmol) and DIPEA (1.6 mL, 9.2 mmol) were added. The white suspension was stirred at rt. The mixture slowly became a light yellow solution over time.

[1444] Acetic acid (786 μL, 13.7 mmol) was added after 1h. The solvent was evaporated, and the product purified by RP-LPLC yielding 23c.

[1445] Yield: 1.77 g (86%)

[1446] MS: m/z 449.15=[M+H].sup.+, (calculated=449.25).

[1447] 23c (1.23 g, 2.74 mmol) and 22b (1.99 g, 2.28 mmol) were dissolved in acetonitrile (53 mL). DMAP (557 mg, 4.56 mmol) was added under stirring and to the resulting solution DIC (1.41 mL, 9.12 mmol) was given. After 1 h 0.7 mL TFA were added and the solvent removed in vacuo. The product was purified by RP-LPLC yielding 23d.

[1448] Yield: 2.33 g (78%, 2× TFA salt)

[1449] MS: m/z 1077.65=[M+H].sup.+, (calculated=1077.57).

[1450] 23d (2.33 g, 1.78 mmol) was dissolved in DCM (10 mL). TFA (10 mL, 131 mmol) was added under stirring. After 45 min the solvent was evaporated and the residue was co-evaporated with 50 mL of DCM. The residue was dried under high vacuum overnight yielding 2.90 g of 23e, which was used without further purification. 23e was dissolved in acetonitrile (68 mL) and 3-maleimidopropionic acid N-hydroxysuccinimide ester (1.19 g, 4.45 mmol) was added under stirring. DIPEA (3.1 mL, 17.8 mmol) was added. After 80 min the reaction was quenched by addition of TFA (1.36 mL, 17.8 mmol). The reaction was concentrated in vacuo to a volume of 40 mL and the product purified by RP-LPLC yielding 23f.

[1451] Yield: 1.73 g (75% over 2 steps, 2× TFA salt)

[1452] MS: m/z 1072.60=[M+H].sup.+, (calculated=1072.49).

[1453] 23f (1.73 g, 1.33 mmol) was dissolved in acetonitrile (17 mL) and EDC (767 mg, 4 mmol), HOSu (462 mg, 4 mmol) and DMAP (19 mg, 0.15 mmol) were added under stirring. After 1.5 h the reaction was quenched by addition of TFA (100 μL, 1.3 mmol) and the reaction was concentrated in vacuo to a volume of 8.5 mL and the product purified by RP-LPLC yielding 23 g.

[1454] Yield: 1.36 g (73%, 2× TFA salt)

[1455] MS: m/z 1169.71=[M+H].sup.+, (calculated=1169.50).

EXAMPLE 19: PREPARATION OF AMINE-HAS 24A AND 24B

[1456] Hyaluronic acid sodium salt (90-130 kDa, 504 mg, 1.26 mmol COOH, 1.00 eq.) was dissolved in 100 mM MES 400 mM 1,3-diaminopropane buffer pH 5.5 (62.5 mL) under vigorous stirring. HOBt (573 mg; 3.74 mmol, 3.00 eq.) and EDC.HCl (223 mg; 1.17 mmol, 0.93 eq.) were added. The suspension was stirred at ambient temperature overnight. Sodium acetate trihydrate (8.48 g) was added, whereupon the suspension turned into a solution. The crude amine-modified HA was precipitated by addition of absolute ethanol, washed with 80% (v/v) ethanol and absolute ethanol and was dried under high vacuum for 1 hour. The pellets were dissolved in water (40 mL) to form a clear solution. 4 M NaOH (13.3 mL) was added and the solution was stirred at ambient temperature for two hours before of acetic acid (3.05 mL) was added. The product was precipitated by addition of absolute ethanol, washed with 80% (v/v) ethanol and absolute ethanol and was dried under high vacuum to give amine-functionalized HA 24a as acetate salt. The amine content of the material was determined by an OPA assay.

[1457] Yield: 432 mg (acetate salt, amine-content: 0.253 mmol/g, 10.4% DS)

[1458] Another amine-HA 24b was prepared analogously to the procedure described above, only using a different amount of EDC.HCl (95.8 mg; 0.50 mmol, 0.404 eq.).

[1459] Yield: 449 mg (acetate salt, amine-content: 0.114 mmol/g, 4.6% DS)

EXAMPLE 20: PREPARATION OF THIOL-HA 25 FROM AMINE-HA 24A

[1460] Amine-functionalized HA 24a (400 mg, 0.101 mmol amines, 1.0 eq.) was dissolved in 100 mM HEPES buffer pH 8.4 (33.25 mL). A freshly prepared solution of SPDP (318 mg, 1.02 mmol, 10.1 eq.) in acetonitrile (18 mL) was added to the mixture while stirring. The mixture was stirred at ambient temperature for 120 minutes before a freshly prepared solution of TCEP (582 mg, 2.03 mmol, 20.1 eq.) in water (5.13 mL) was added to the reaction mixture. The solution was stirred for one hour at ambient temperature before 1 M sodium acetate buffer pH 5.5 (56.4 mL) was added. The product was collected by addition of absolute ethanol and centrifugation. After washing with 80% (v/v) ethanol, absolute ethanol and drying in high vacuum for five hours, crude thiol-HA was obtained as white solid. The crude material was dissolved in 1% acetic acid (40 mL) by vigorous stirring under an argon atmosphere. 1 M sodium acetate buffer pH 5.5 (40 mL) was added to the solution and the resulting mixture was filtered through a 0.22 μm PES bottle-top filter. The product was precipitated from the filtrate by addition of absolute ethanol and centrifugation. After washing with 80% (v/v) ethanol and absolute ethanol, the material was dried in high vacuum for six hours to give thiol-HA 25 as off-white pellets. Thiol content was determined via Ellman assay.

[1461] Yield: 366 mg (thiol-content: 0.209 mmol/g)

EXAMPLE 21: PREPARATION OF MALEIMIDE-HA 26 FROM AMINE-HA 24B

[1462] Amine-functionalized HA 24b (443 mg, 0.05 mmol amines, 1.0 eq.) was dissolved in 100 mM HEPES buffer pH 7.4 (44.25 mL). A freshly prepared solution of 3-maleimidopropionic acid NHS ester (134 mg, 0.49 mmol, 10.0 eq.) in acetonitrile (9.7 mL) was added to the mixture while stirring. The mixture was stirred at ambient temperature for 60 minutes before 1 M sodium acetate buffer pH 5.5 (54 mL) was added. The product was collected by addition of absolute ethanol and centrifugation. After washing with 80% (v/v) ethanol, followed by washing with absolute ethanol, the material was stored at −20° C. overnight and was dried in high vacuum for two hours the next day to yield crude maleimide-HA as white solid. The crude material was dissolved in 1% acetic acid (44.25 mL) by vigorous stirring. 1 M sodium acetate buffer pH 5.5 (54 mL) was added to the solution and the resulting mixture was filtered through a 0.22 μm PES bottle-top filter. The product was precipitated from the filtrate by addition of absolute ethanol and centrifugation. After washing with 80% (v/v) ethanol and absolute ethanol, the material was dried in high vacuum for six hours to give maleimide-HA 26 as white pellets. Maleimide content was determined via reverse-Ellman assay.

[1463] Yield: 376 mg (maleimide-content: 0.109 mmol/g)

EXAMPLE 22: PREPARATION OF CROSSLINKED HA MICROPARTICLES WITH FREE THIOLS 27

[1464] Thiol-HA 25 (90.5 mg) was dissolved in 200 mM MES, 3 mM EDTA buffer pH 5.5 (3015 μL) by vigorous shaking under an argon atmosphere to produce a 30 mg/mL solution of the compound in buffer (solution A). Maleimide-HA 26 (70.7 mg) was dissolved in 200 mM MES, 3 mM EDTA buffer pH 5.5 (2355 μL) by vigorous shaking to produce a 30 mg/mL solution of the compound in buffer (solution B). In a 2 mL Eppendorf tube, equipped with a magnetic stirring bar, 200 mM MES, 3 mM EDTA buffer pH 5.5 (94.2 μL) was mixed with solution A (717.7 μL) and solution B (688.1 μL) under vigorous shaking. For gelling, the mixture was left standing at r.t. under an argon atmosphere overnight. The gel was transferred into a 5 mL Luer-Lock syringe to which a line of a male/female Luer Lock adapter, a 2×1 mm PTFE o-ring, a 144 μm stainless steel mesh (4 mm diameter), a 2×1 mm PTFE o-ring, a male/female Luer Lock adapter, a 2×1 mm PTFE o-ring, a 144 μm stainless steel mesh (4 mm diameter), a 2×1 mm PTFE o-ring and a male/female Luer Lock adapter was connected. The gel portion in the syringe was passed through the two 144 μm stainless steel meshes into 200 mM MES, 3 mM EDTA buffer pH 5.50 in a 15 mL Falcon tube. The hydrogel was successively washed with 3 mM EDTA buffer pH 5.5 followed by 200 mM succinate, 3 mM EDTA buffer pH 4.0, and 200 mM succinate, 3 mM EDTA, 0.5% Tween 20 buffer pH 4.0 by shaking, centrifugation and supernatant removal. After the last washing step, the volume of the gel suspension was adjusted to 10 mL with 3 mM EDTA, 0.5% Tween 20 buffer pH 4.0 in a 15 mL Falcon tube to yield the cross-linked HA with free thiol groups as colorless and almost completely transparent suspension. The thiol content of the hydrogel suspension was determined by Ellman assay.

[1465] Yield: 10 mL

[1466] Hydrogel content: 4.2 mg/mL (nominal, not experimentally determined)

[1467] Thiol content (suspension, fresh): 192 μmol/L

[1468] After 4 weeks storage at 5° C. under an argon atmosphere, the thiol content of the hydrogel suspension 27 and the particle-free supernatant of the latter after thorough centrifugation was determined by Ellman assay.

[1469] Thiol content (suspension, 4 weeks): 184 μmol/L

[1470] Thiol content (supernatant, 4 weeks): 1 μmol/L

EXAMPLE 23: PREPARATION OF CTLA-4 MAB-LINKER CONJUGATE MIXTURE 28

[1471] 204.13 mL of CTLA-4 mAB at 5.341 mg/mL in 26 mM Tris-HCl, 100 mM NaCl, 55 mM mannitol, 0.1 mM pentetic acid (DTPA), 0.01% Tween80, pH 7.0 was used in this example. CTLA-4 mAB was buffer exchanged to 30 mM sodium phosphate, pH 7.4, concentrated, and the protein concentration was adjusted to nominal 10 mg/mL. 103.14 mL CTLA-4 mAB in 30 mM sodium phosphate, pH 7.4 at a concentration of 9.74 mg/mL were prepared.

[1472] 3 mol eq. (218.6 μL) of linker reagent 23 g (corrected with respect to NHS content, 100 mM stock solution in DMSO) relative to the amount of CTLA-4 mAB were added to the protein solution. The reaction mixture was mixed carefully and incubated for 5 min at ambient temperature yielding a mixture of unmodified CTLA-4 mAB and the protected CTLA-4 mAB-linker conjugates (e.g. monoconjugate, bisconjugate) 28a.

[1473] The conjugation reaction was immediately followed by a pH shift towards about pH 4 and a cation exchange chromatography (CIEC) step was performed to remove excess linker species from the CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28. The pH shift was achieved by addition of 0.12 vol. eq. (12.4 mL) of 0.5 M succinic acid, pH 3.0 with respect to the volume of the CTLA-4 mAB solution (103.1 mL), and the solution was mixed carefully. The CIEC step was performed using an Aekta pure system equipped with an Eshmuno CPX column (8 mm ID×200 mm length, CV=10 mL) with 20 mM succinic acid, pH 5.5 as mobile phase and a linear salt gradient elution with sodium chloride (0-60% 20 mM succinic acid, 1 M NaCl, pH 5.5 in 15 CV) at a flow rate of 4.0 mL/min. Three runs with ˜39 mL injection volume (˜337 mg) per run were performed and 119.27 mL of CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture was collected at a concentration of 7.32 mg/mL. The collected CIEC fractions were analyzed by HPLC-MS to confirm the removal of unreacted, free linker reagent 23 g.

[1474] To determine the content of protected CTLA-4 mAB-linker mono-, bis-, and tris-conjugate within CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28, a PEGylation with 20 kDa PEG thiol and subsequent SE-HPLC analysis was performed.

[1475] 20 μL unmodified CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28 (c=7.32 mg/mL) were adjusted to 5 mg/mL by the addition of 9.28 μL of 20 mM succinic acid, pH 5.5 immediately followed by the addition of 1/19 vol. eq. 20 mM succinic acid, 100 mM EDTA, 0.2% Tween20, pH 5.5 (1.5 μL) with respect to the volume of 29.3 μL. 4.6 mg of 20 kDa PEG thiol were dissolved in water (115 μL) and 10 mol. eq. relative to the amount of CTLA-4 mAB were added (5 μL). After 45 minutes incubation at ambient temperature, SE-HPLC was performed using an Agilent 1200 system connected to a Tosoh TSKgel UP-SW3000 column with 100 mM KH.sub.2PO.sub.4, 100 mM Na.sub.2SO.sub.4, pH 6.7 as mobile phase. A total maleimide content of 41% was determined for the CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28.

[1476] After analysis and overnight storage at 4° C., 118.38 mL of the CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28 (c=7.32 mg/mL) were adjusted to a final concentration of 5 mM EDTA and 0.01% Tween20 with 1/19 vol. eq. of 20 mM succinic acid, 100 mM EDTA, 0.2% Tween20, pH 5.5 (6.2 mL) with respect to the volume of 118.38 mL and the solution was shaken carefully. The sample was filtered using one qpore Plastic vacuum filter (PVDF membrane) with a pore size of 0.22 μm. 122.67 mL of the adjusted CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28 at a concentration of 7.82 mg/mL was obtained.

EXAMPLE 24: SYNTHESIS OF TRANSIENT CTLA-4 MAB-LINKER-HYDROGEL PRODRUG 29B AND 29C

[1477] Conjugation of CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28 to thiol functionalized, crosslinked HA hydrogel 27 was performed by addition of CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28 to 1.5 mol. hydrogel 27 with respect to determined total maleimide content of 41% (4 μM) as described in example 23 in the CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28.

[1478] 7.5 mL of hydrogel suspension prepared according to example 22 (4.22 mg/mL nominal gel content with a thiol content of 200.8 μM) in 20 mM succinic acid, 150 mM NaCl, 3 mM EDTA, 0.1% Tween20, pH 4.0 were transferred into a 15 mL Falcon tube. Four 15 mL Falcon tubes were prepared in total. The hydrogel particles were sedimented by centrifugation at 4000 rcf for 1 minute and the supernatant was removed by pipetting. Washing of the particles was accomplished via five cycles of washing steps, which included addition of 10 mL 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5 buffer, centrifugation at 1000 rcf for 1 minute and careful removal of the supernatant by pipetting. After the last washing step, each of the four falcon tubes was filled up to a nominal total volume of suspension of 4 mL with above mentioned buffer. 2.6 mL (nominal) of the hydrogel suspension were transferred into a separate 50 mL Falcon tube resulting in four Falcon tubes each containing nominal 2.6 mL of washed hydrogel suspension.

[1479] 122.62 mL of the adjusted CTLA-4 mAB/protected CTLA-4 mAB-linker conjugate mixture 28 (c=7.82 mg/mL, 958.3 mg) at pH 5.5 were divided in four parts and approx. 33 mL were added to each of the four 50 mL Falcon tubes containing the hydrogel suspension described above. The resulting suspensions were mixed end-over-end and incubated at ambient temperature under gentle agitation overnight yielding protected transient CTLA-4 mAB-linker hydrogel prodrug 29a.

[1480] The protected transient CTLA-4 mAB-linker hydrogel prodrug 29a was sedimented by centrifugation at 1000 ref for 1 minute and resting for 3 minutes. The supernatants after the hydrogel loading were transferred in a 250 mL Corning bottle by pipetting. The protected transient CTLA-4 mAB-linker hydrogel prodrug 29a were combined in one 50 mL Falcon tube.

[1481] For blocking of the remaining unreacted thiols, the protected transient CTLA-4 mAB-linker hydrogel prodrug 29a was first washed seven times with 30 mL 10 mM iodoacetamide (IAA) in 30 mM sodium phosphate, 50 mM N,N,N′-Trimethylethylendiamine (TriMED), 0.01% Tween20, pH 7.4. Afterwards, 30 mL 10 mM IAA in 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4 were added to the sedimented protected transient CTLA-4 mAB-linker hydrogel prodrug 29a and incubated at ambient temperature under gentle agitation for 1 h. Removal of IAA blocking solution was accomplished via ten cycles of washing, which included addition of 30 mL 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4 buffer, centrifugation at 1000 rcf for 1 minute and careful removal of the supernatant by pipetting after 3 minutes resting.

[1482] Afterwards, for deprotection of the protected transient CTLA-4 mAB-linker hydrogel prodrug 29a, 30 mL 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4 buffer were added to the sedimented hydrogel and the resulting suspension was incubated at 25° C. overnight yielding transient CTLA-4 mAB-linker hydrogel prodrug 29b. Final formulation of transient CTLA-4 mAB-linker hydrogel prodrug 29b was performed by washing the transient CTLA-4 mAB-linker hydrogel prodrug 29b ten times with 20 mM succinic acid, 10 w % a-a-D-trehalose, 0.01% Tween20, pH 5.5. 2.5 mL of 29b were diluted with 10 mL of 20 mM succinic acid, 10 w % α-α-D-trehalose, 0.01% Tween20, pH 5.5 to give 29c.

EXAMPLE 25: IN VITRO RELEASE KINETICS FOR 29B

[1483] 25 mg of dense CTLA-4 mAB-linker hydrogel prodrug 29b (corresponding to approximately 0.45 mg protein) were transferred in a sterile, 1.5 mL Eppendorf tube. Eight tubes were prepared in total. 1 mL 60 mM sodium phosphate, 3 mM EDTA, 0.01% Tween20, pH 7.4 was added to each tube, which was subsequently mixed end-over-end and incubated without agitation for 5 minutes. The supernatant was removed to a final volume of 0.5 mL suspension per vial. The suspensions were incubated at 37° C. in a water bath. After different time intervals, one vial was removed from 37° C., centrifuged and the supernatant was analyzed by A280 measurement and SE-HPLC at 215 nm. The relative amount of released CTLA-4 mAB based on concentration determination of the supernatant with respect to the total amount of CTLA-4 mAB in each vial was plotted against the incubation time in days.

[1484] Release Kinetics from 29b:

TABLE-US-00003 CTLA-4 mAB CTLA-4 mAB t [d] release [μg] release [%] 1.92 25.9 5.5 8.92 83.2 18.5 18.92 149.9 33.0 27.92 189.4 42.9 39.92 226.2 49.2

EXAMPLE 26: SYNTHESIS OF PLACEBO HYDROGEL 30

[1485] 16.7 mL of hydrogel suspension 27 were distributed over four 15 mL Falcon tubes (4.16 mL each). The tubes were briefly centrifuged, and the volume of the suspension was reduced to 3 mL by partial removal of the supernatant. 10 mL 10 mM iodoacetamide (IAA) in 30 mM sodium phosphate, 50 mM N,N,N′-Trimethylethylendiamine, 0.01% Tween20, pH 7.4 (blocking solution) were added into each of the four Falcon tubes. The suspension was gently mixed and briefly centrifuged. 10 mL of the supernatant were discarded. This procedure was performed overall 7 times. 10 mL of the blocking solution were added into each of the 4 Falcon tubes. The suspension was gently mixed, and the resulting suspension was incubated at ambient temperature for 60 min. The suspension was gently mixed and briefly centrifuged. 10 mL of the supernatant were discarded. The hydrogel suspension was washed 10 times with 20 mM succinic acid, 10 wt % α-α-D-trehalose, 0.01% Tween20, pH 5.5. For this purpose, 10 mL buffer were added, and the suspension was gently mixed and briefly centrifuged. 10 mL of the supernatant were discarded.

[1486] After the tenth washing cycle, 5 mL of fresh buffer were added to two of the four tubes and the suspension was mixed well. The resulting suspensions were each transferred into another Falcon tube in which hydrogel suspension without fresh buffer was present. The suspensions were mixed well and briefly centrifuged. The supernatant was reduced to an overall volume of 6 mL and the suspensions were combined in one single Falcon tube. The resulting suspension was briefly centrifuged, and the volume of the suspension was reduced to 11 mL to give the placebo hydrogel 30 with an approximate hydrogel content of 7.1 mg/mL.

EXAMPLE 27: PLASMA PHARMACOKINETICS OF CTLA-4 MAB IN WISTAR RATS AFTER SUBCUTANEOUS (SC) INJECTIONS OF A TRANSIENT CTLA-4 MAB-LINKER-HYDROGEL PRODRUG (COMPOUND 29B) AND AFTER INTRAVENOUS (IV) AND SUBCUTANEOUS (SC) INJECTIONS OF FREE CTLA-4 MAB

[1487] This study was performed in order to investigate the plasma pharmacokinetics of CTLA-4 mAB in Wistar rats following subcutaneous administration of transient CTLA-4 mAB-linker-hydrogel prodrug 29b or following subcutaneous or intravenous administration of free CTLA-4 mAB. Animals (n=3 per group) received either a single SC injection of a 29b (1 mg/kg CTLA-4 mAB equivalents) in the neck region or a single SC injection in the neck region or IV injection in the tail vein of an CTLA-4 mAB formulation (1 mg/kg CTLA-4 mAB). At selected time points, 200 μL blood were collected in L.sup.1-Heparin tubes and processed to plasma by centrifugation at 3,000 g for 10 minutes at 4° C.

[1488] CTLA-4 mAB concentrations in rat plasma were determined with a commercial ELISA setup obtained from BioVision Inc. (CA, USA, order number E4384-100). The manufacturer's instructions were followed with minor changes to the protocol. Specifically, the plasma samples were used undiluted (except for samples above the upper limit of quantification which were diluted with blank plasma prior to the measurement) and the sample incubation time was prolonged to 60 minutes.

[1489] Calibration standards of CTLA-4 mAB in blank plasma were prepared as follows: thawed L.sup.1-Heparin Wistar rat plasma was homogenized. The free CTLA-4 mAB formulation was spiked into blank plasma at concentrations between 5,000 ng/mL and 50 ng/mL with additional higher and lower anchor points. These solutions were used for the generation of a calibration curve. Calibration curves were analyzed via a 4-parameter logarithmic fit and 1/Y.sup.2 weighted. The determined CTLA-4 mAB plasma concentrations are depicted in Table 1.

TABLE-US-00004 TABLE 1 Determined mean CTLA-4 mAB plasma concentrations in ng/mL per time point and group (n = 3). Time (h) Group 1 4 24 32 48 72 96 168 1 <LLOQ <LLOQ 170 240 370 560 650 780 2 170 600 3700 4800 6200 6900 6100 5700 3 18000 14000 9600 7500 6000 4900 4000 3100 Group 1: transient CTLA-4 mAB-linker-hydrogel prodrug (compound 29b; 1 mg/kg CTLA-4 mAB equivalents - subcutaneous administration); Group 2: CTLA-4 mAB (1 mg/kg - subcutaneous administration); Group 3: CTLA-4 mAB (1 mg/kg - intravenous administration); LLOQ at 50 ng/mL

[1490] Specifically, CTLA-4 mAB concentration after intra-tissue (subcutaneous) injection of a transient CTLA-4 mAB-linker-hydrogel prodrug (compound 29b; 1 mg/kg) were below 1 μg/mL 72 h after administration.

[1491] Also, CTLA-4 mAB concentration 72 h after intra-tissue (subcutaneous) injection of a transient CTLA-4 mAB-linker-hydrogel prodrug (compound 29b; 1 mg/kg) were at least 80% of CTLA-4 mAB concentration 1 h after intra-tissue (subcutaneous) injection of a transient CTLA-4 mAB-linker HA-hydrogel conjugate (compound 29b; 1 mg/kg).

[1492] In addition, CTLA-4 mAB concentration 24 h after intra-tissue (subcutaneous) injection of transient CTLA-4 mAB-linker-hydrogel prodrug (compound 29b; 1 mg/kg) were at least 50% lower than CTLA-4 mAB concentration 24 h after intra-tissue (subcutaneous) of an CTLA-4 mAB formulation (1 mg/kg CTLA-4 mAB).). In fact, levels of CTLA-4 mAB concentration 24 h after intra-tissue (subcutaneous) injection of transient CTLA-4 mAB-linker-hydrogel prodrug (compound 29b; 1 mg/kg) were more than 90% lower than CTLA-4 mAB concentration 24 h after intra-tissue (subcutaneous) injection of an CTLA-4 mAB formulation (1 mg/kg CTLA-4 mAB). This is significant and noteworthy as higher exposure of CTLA4 mAb in patients is significantly associated with higher rates of adverse events clinically (Feng et al. Exposure-Response Relationships on the Efficacy and Safety of Ipilimumab in Patients with Advanced Melanoma.” Clinical Cancer Research. 2013. 19 (14); 3997-86).

[1493] In addition, CTLA-4 mAB plasma concentrations after intra-tissue (subcutaneous) injection of transient CTLA-4 mAB-linker-hydrogel prodrug (compound 29b; 1 mg/kg) were substantially lower than CTLA-4 mAB concentrations from intravenous injection of an CTLA-4 mAB formulation (1 mg/kg CTLA-4 mAB) at every timepoint measured such as 56.5 fold lower at 24h, 31.3 fold lower at 32h, 16.2 fold lower at 48h, 8.8 fold lower at 72h, 6.2 fold lower at 96h and 4.0 fold lower even after 168h. This is significant and noteworthy as higher exposure of CTLA4 mAb in patients is significantly associated with higher rates of adverse events clinically (Feng et al. Exposure-Response Relationships on the Efficacy and Safety of Ipilimumab in Patients with Advanced Melanoma.” Clinical Cancer Research. 2013. 19 (14); 3997-86).

EXAMPLE 28: IN VIVO ANTI-TUMOR EFFICACY

[1494] The study was conducted in female C57BL/6 mice with the human CTLA-4 gene knocked into the endogenous CTLA-4 locus with an age of 6-11 weeks at the day of tumor inoculation. This model is also known as a Human Genetically Engineered Mouse Model or HuGEMM. Mice were subcutaneously implanted with 1×10.sup.6 MC38 tumor cells in the right flank. When tumors to be injected were grown to a mean tumor volume of ˜65 mm.sup.3, mice were randomized into treatment cohorts (day 0) and treated with either: 1) a single 50 μL intratumoral injection of control hydrogel 30 or 2) a single 50 μL intratumoral injection of 840 μg of CTLA-4 mAB-linker-hydrogel prodrug 29b or 3) four total 200 μL intraperitoneal doses of 210 μg given on Days 0, 4, 8, and 12 of CTLA-4 mAB. Hydrogels were administered as suspensions in 20 mM succinate, 135 mM NaCl, 0.01% Tween-20, pH 4.0 buffer. Following treatment initiation, anti-tumor efficacy was assessed by determination of tumor volumes at various time points from tumor size measurements with a caliper. Tumor volumes were calculated according to the formula:

[1495] Tumor volume=(L×W.sup.2)×0.5 where L is the length of the tumor and W the width (both in mm). Mice were removed from the study once tumors were greater than 3000 mm.sup.3.

[1496] Results: Significant tumor growth inhibition was observed with either systemically delivered free CTLA-4 mAB (CTLA-4 mAB IP) or intratumorally delivered CTLA4 mAb Hydrogel 29b (CTLA4 mAb Hydrogel IT 29b) as compared to treatment with intratumorally delivered control hydrogel (control hydrogel 30) with respective average tumor volumes at day 20 post initiation of dosing of 60.5, 203.9, and 2142.5 mm.sup.3 respectively (Table 2). One-way ANOVA demonstrated that CTLA-4 mAB IP or CTLA-4 mAB hydrogel 29b IT treatment was statistically significant vs. control hydrogel 30 IT treatment (p values of <0.0001 for both treatments) with no significant difference between CTLA-4 mAB IP systemic or CTLA-4 mAB hydrogel 29b IT treatment (p=0.9068, Table 2).

TABLE-US-00005 TABLE 2 Summary of CTLA4 treatment efficacy results in MC38 tumor bearing mice Mean SEM P-value* P-value* Tumor Tumor vs vs Volume Volume Control CTLA4 (mm3) (mm3) N Hydrogel Ab IP Group Day 20 Day 20 Day 20 Overall Overall Control 2142.5 406.7 8 NA <0.0001 hydrogel 30 CTLA-4 mAB IP 60.5 10.2 8 <0.0001 NA CTLA-4 mAB 203.9 56.1 8 <0.0001  0.9046 Hydrogel 29b IT SEM = standard error of the mean, N = sample size; *Significance was determined by One-way ANOVA followed by multiple comparisons using Tukey's Honest Significant Differences (HSD) post-hoc test.

EXAMPLE 29: PREPARATION OF HHC.SUP.MET.-LINKER CONJUGATE MIXTURE 31

[1497] 154 mL of HHC.sup.MET at 5.94 mg/mL in PBS, pH 7.4 was used in this example. HHC.sup.MET was concentrated using centrifugal filters, and the protein concentration was determined. 28.18 mL HHC.sup.MET in PBS, pH 7.4 at a concentration of 30.3 mg/mL were prepared.

[1498] 1.5 mol eq. (508 μL) of linker reagent 23 g (corrected with respect to NHS content, 100 mM stock solution in DMSO) relative to the amount of HHC.sup.MET were added to the protein solution. The reaction mixture was mixed carefully and incubated for 5 min at ambient temperature yielding a mixture of unmodified HHC.sup.MET and the protected HHC.sup.MET conjugates (e.g. monoconjugate, bisconjugate) 31.

[1499] The linker-conjugation reaction was immediately followed by a pH shift towards about pH 4 and a buffer exchange was performed to remove excess linker species from the HHC.sup.MET/HHC.sup.MET-linker conjugate mixture 31. The buffer shift was achieved by addition of 0.047 vol. eq. (1.324 mL) of 0.4 M succinic acid, pH 3.0 with respect to the volume of the HHC.sup.METsolution (28.18 mL), and the solution was mixed carefully end-over-end. The buffer exchange to 20 mM succinic acid, pH 4.0 was performed using an Äkta purifier 100 system equipped with a GE HiPrep column at a flow rate of 8.0 mL/min. Six runs with approx. 5 mL injection volume per run were performed.

[1500] To determine the content of protected HHC.sup.MET-linker mono-, bis-, and tris-conjugate within HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 31, a PEGylation with 20 kDa PEG thiol and subsequent SE-HPLC analysis was performed. 24.4 μL unmodified HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 31 (c=10.89 mg/mL) were pH-adjusted to pH 5.5 by addition of 0.154 vol. eq. (3.8 μL) of 0.5 M succinic acid, pH 6.2 with respect to the volume of the HHC.sup.MET solution (24.4 μL). The obtained solution was then supplemented with 1/19 vol. eq. 20 mM succinic acid, 100 mM EDTA, 0.2% Tween20, pH 5.5 (1.5 μL) with respect to the volume of 28.2 μL. The protein concentration of the unmodified HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 31 at pH 5.5 was adjusted to 4 mg/mL by mixing 15.8 μL of the solution with 20.2 μL of 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5. The PEGylation reaction was started by the addition of 4 μL of 15 mM PEG20-SH solution in water. After 15 minutes incubation at ambient temperature, SE-HPLC was performed using an Agilent 1200 system connected to a Superdex 200 Increase 10/300 GL column with PBS-T, pH 7.4 as mobile phase. Maleimide content was calculated with the use of the peak area of the conjugates and multiplied with the number of attached PEG reagents. A total maleimide content of 47.7% was determined for the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 31.

[1501] After analysis and overnight storage at 4° C., 71.98 mL of the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 31 (c=10.89 mg/mL) were pH-adjusted to pH 5.5 by addition of 0.154 vol. eq. (11.08 mL) of 0.5 M succinic acid pH 6.2. The obtained solution was supplemented with 1/19 vol. eq. 20 mM succinic acid, 100 mM EDTA, 0.2% Tween20, pH 5.5 (4.37 mL) and the solution was mixed end-over-end. The sample was filtered using one qpore Plastic vacuum filter (PVDF membrane) with a pore size of 0.22 μm.

EXAMPLE 30: SYNTHESIS OF TRANSIENT HHC.SUP.MET.-LINKER-HYDROGEL PRODRUG 32

[1502] Conjugation of HHC.sup.MET/HHC.sup.MET-linker conjugate mixture 31 to the reduced thiol functionalized hydrogel 19 was performed by addition of HHC.sup.MET/HHC.sup.MET-linker conjugate mixture 31 to 1.75 mol. eq. of thiol groups in hydrogel 19 with respect to determined total maleimide content of 47.7% (19.13 μmol) as described in example 29 in the HHC.sup.MET/HHC.sup.MET-linker conjugate mixture 31.

[1503] 8.5 mL of MTS functionalized hydrogel 18 (23.7 mg/mL nominal gel content with a thiol content of 0.183 mmol/g) in 20 mM succinic acid, 0.01% Tween20, pH 4.0 were transferred into a 20 mL syringe with a frit. The thiol functionalized hydrogel was reduced by replacement of the storage solution by 20 mL of 50 mM TCEP solution in PBS-T and incubation for 15 minutes at ambient temperature. Afterwards, the 50 mM TCEP solution was removed from the syringe, and the hydrogel was washed in the syringe 10 times with 20 mL 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5 and resuspended in ˜ 6.7 mL of 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5 to yield 19.

[1504] 3.06 mL of hydrogel 19 were transferred into two 50 mL falcon tubes. Afterwards, 43.2 mL of the HHC.sup.MET/protected HHC.sup.MET-linker conjugate mixture 31 (c=9.26 mg/mL) at pH 5.5 were added into each falcon tube containing hydrogel 19. The resulting suspensions were mixed well and incubated at ambient temperature under gentle rotation overnight yielding protected transient HHC.sup.MET-linker hydrogel prodrug.

[1505] After overnight incubation, the protected transient HHC.sup.MET-linker hydrogel prodrug was transferred into a 20 mL syringe equipped with a frit, and washed in the syringe once with 20 mL 20 mM succinic acid, 5 mM EDTA, 0.01% Tween20, pH 5.5 and two times with 20 mL 10 mM iodoacetamide in 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4. The protected transient HHC.sup.MET-linker hydrogel prodrug was incubated for 60 minutes with gentle rotation in 30 mM sodium phosphate, 10 mM iodoacetamide, 50 mM TriMED, 0.010% Tween20, pH 7.4 buffer in the syringe at ambient temperature. After, the hydrogel was washed ten times in the syringe with 20 mL 30 mM sodium phosphate, 200 mM TriMED, 0.01% Tween20, pH 7.4. The solvent was each time discarded.

[1506] 20 mL 30 mM sodium phosphate, 200 mM TriMED, 0.01% Tween20, pH 7.4 buffer were drawn up into the syringe and the resulting suspension was incubated at 25° C. for 26 hours under gentle rotation yielding transient HHC.sup.MET-linker hydrogel prodrug 32. Formulation of transient HHC.sup.MET-linker hydrogel prodrug 32 was achieved by washing the hydrogel ten times in the syringe with 20 mL 20 mM succinic acid, 8.5% α-α-D-trehalose, 1% carboxymethylcellulose, 0.01% Tween20, pH 5.0.

EXAMPLE 31: SYNTHESIS OF TRANSIENT CTLA-4 MAB-LINKER-HYDROGEL PRODRUG 33

[1507] Preparation of the transient CTLA-4 mAB-linker hydrogel prodrug was performed as described in example 24 yielding transient CTLA-4 mAB-linker hydrogel prodrug 29b. However, following the overnight incubation in 30 mM sodium phosphate, 50 mM TriMED, 0.01% Tween20, pH 7.4 buffer, the transient CTLA-4 mAB-linker hydrogel prodrug was washed ten times with 20 mM succinic acid, 135 mM NaCl, 1 w % carboxymethylcellulose (CMC), 0.01% Tween20, pH 4.0 instead of 20 mM succinic acid, 10 w % α-α-D-trehalose, 0.01% Tween20, pH 5.5 for final formulation to give 33.

EXAMPLE 32: PLASMA PHARMACOKINETICS OF HHC.SUP.MET .IN WISTAR RATS AFTER SUBCUTANEOUS (SC) AND INTRAMUSCULAR (IM) INJECTIONS OF A TRANSIENT HHC.SUP.MET.-LINKER HYDROGEL PRODRUG 32 AND AFTER INTRAVENOUS (IV) AND SUBCUTANEOUS (SC) INJECTIONS OF FREE HHC.SUP.MET

[1508] This study was performed in order to investigate the plasma pharmacokinetics of HHC.sup.MET in Wistar rats following subcutaneous and intramuscular administration of transient HHC.sup.MET-linker hydrogel prodrug 32 or following subcutaneous or intravenous administration of free HHC.sup.MET. Animals (n=3 per group) received either a single SC injection in the neck region or a single IM injection in the thigh musculature of a formulation of 32 (10 mg/kg HHC.sup.MET equivalents) or a single SC injection in the neck region or IV injection in the tail vein of an HHC.sup.MET formulation (10 mg/kg HHCMet). At selected time points, 200 μL blood were collected in L.sup.1-Heparin tubes and processed to plasma by centrifugation at 3,000 g for 10 minutes at 4° C.

[1509] HHC.sup.MET concentrations in rat plasma were determined with an in-house developed sandwich ELISA setup. For capturing HHC.sup.MET, a human CTLA-4 (AA Ala37-Ser160)—Fc Tag fusion protein (Supplier AcroBiosystem, Newark, Del.; USA, catalog no. CT4-H5255) was coated to the ELISA plate wells and read-out was performed via a rabbit anti-camelid VHH antibody conjugated with horseradish peroxidase (supplier Genscript, Piscataway, N.J., USA, catalog no. A01861-200).

[1510] Calibration standards of HHC.sup.MET in blank plasma were prepared as follows: thawed L.sup.1-Heparin Wistar rat plasma was homogenized. The free HHC.sup.MET formulation was spiked into blank plasma at concentrations between 96.0 ng/mL and 3.00 ng/mL with additional higher and lower anchor points. These solutions were used for the generation of a calibration curve. Calibration curves were analyzed via a 4-parameter logarithmic fit and 1/Y weighted. Calibration curves were confirmed via separately prepared quality control standards at 10, 40 and 80 ng/mL.

[1511] The determined HHC.sup.MET plasma concentrations are depicted in Table 3.

TABLE-US-00006 TABLE 3 Determined mean HHC.sup.MET concentrations in ng/mL per time point and group (n = 3). Time (h) Group 0.25 1 4 24 32 48 72 96 168 1 — 5.40 30.2 52.9 39.7 41.2 33.7 34.8 38.7 2  2540 5090 2280 2.60 <LLOQ <LLOQ 2.07 <LLOQ <LLOQ 3 17400 3530 1720 8.27 <LLOQ <LLOQ 1.15 <LLOQ <LLOQ 4 — 5.09 27.5 55.3 54.4 46.2 37.1 38.9 56.4 Group 1: Transient HHC.sup.MET-linker hydrogel prodrug 32 (10 mg/kg HHC.sup.MET equivalents - subcutaneous administration); Group 2: HHC.sup.MET (10 mg/kg - subcutaneous administration); Group 3: HHC.sup.MET (10 mg/kg - intravenous administration); Group 4: Transient HHC.sup.MET-linker hydrogel prodrug 32 (10 mg/kg HHC.sup.MET equivalents - intramuscular administration); method LLOQ at 3.00 ng/mL; ,, —″ denotes sample not taken

[1512] Specifically, HHC.sup.MET concentration 72 h after intra-tissue (subcutaneous or intramuscular) injection of transient HHC.sup.MET-linker hydrogel prodrug 32 (10 mg/kg HHC.sup.MET equivalents) is at least 80% of HHC.sup.MET concentration 1 h after intra-tissue (subcutaneous or intramuscular) injection of transient HHC.sup.MET-linker hydrogel prodrug 32 (10 mg/kg HHC.sup.MET equivalents).

EXAMPLE 33: IN VIVO ANTI-TUMOR EFFICACY

[1513] The study was conducted in female C57BL/6 mice with the human CTLA4 gene knocked into the endogenous CTLA4 locus with an age of 6-11 weeks at the day of tumor inoculation. This model is also known as a Human Genetically Engineered Mouse Model or HuGEMM. Mice were subcutaneously implanted with 1×10.sup.6 MC38 tumor cells in the right flank. When tumors to be injected were grown to a mean tumor volume of ˜65 mm.sup.3, mice were randomized into treatment cohorts (day 0) and treated with either: 1) a single 50 μL intratumoral injection of control hydrogel 30 or 2) four total 200 μL intraperitoneal doses of 18 μg given on days 0, 4, 8, and 12 of free CTLA-4 mAB, or 3) a single 50 μL intratumoral injection of 840 μg of CTLA-4 mAB-linker-hydrogel prodrug 29b or 3) a single 50 μL intratumoral injection of 72 μg of CTLA-4 mAB-linker-hydrogel prodrug 29c. Hydrogels were administered as suspensions in 20 mM succinate, 135 mM NaCl, 0.01% Tween20, pH 4.0 buffer. Following treatment initiation, anti-tumor efficacy was assessed by determination of tumor volumes at various time points from tumor size measurements with a caliper. Tumor volumes were calculated according to the formula: Tumor volume=(L×W.sup.2)×0.5 where L is the length of the tumor and W the width (both in mm). Mice were removed from the study once tumors were greater than 3000 mm.sup.3. Percent tumor growth inhibition (TGI) was calculated according to the formula: [1−(Mean Tumor Volume in treated animals)/(Mean Tumor Volume in control animals)]*100.

[1514] Results: Intratumorally delivered control Hydrogel 30 resulted in an average tumor volume of 2142.5 mm.sup.3 at day 20 post initiation of dosing. As compared to 30, significantly lower tumor sizes were observed with either systemically delivered free CTLA-4 mAB (IP) or intratumorally delivered 29b resulting in average tumor volumes of 57.1 and 203.9 mm.sup.3, respectively and percent TGI values of 97.3%, 90.5%, respectively at day 20 post initiation of dosing (Table 4). One-way ANOVA demonstrated that free CTLA-4 mAB and 29b treatment were statistically significant vs 30 treatment (p-values of 0.0014 and 0.0008, respectively) with no significance comparing free CTLA-4 mAB and 29b treatment (p=0.9906, Table 4). A non-significant decrease in tumor growth was seen in the 29c treated animals (average tumor volume: 1580.2 mm.sup.3, 26.2% TGI)

TABLE-US-00007 TABLE 4 Summary of CTLA4 treatment efficacy results in MC38 tumor bearing mice at Day 20 Mean SEM P-value* Percent Tumor Tumor P-value* vs G3: Tumor Volume Volume vs G1: Free CTLA4 Growth Group (mm3) (mm3) N Control Ab IP Inhibition Control hydrogel 30 2142.5 406.7 8 NA 0.0014 NA CTLA-4 mAB 57.1 3.7 5 0.0014 NA 97.3% 29b 203.9 56.1 8 0.0008 0.9906 90.5% 29c 1580.2 405.4 8 0.5700 0.0233 26.2% SEM = standard error of the mean, N = sample size; *Significance between Mean Tumor Volumes was determined by One-way ANOVA followed by multiple comparisons using Tukey's Honest Significant Differences (HSD) post-hoc test.

EXAMPLE 34: FLOW CYTOMETRIC PROFILING OF BLOOD, SPLEEN AND TUMOR IMMUNE CELLS

[1515] The study was conducted in female C57BL/6 mice with the human CTLA4 gene knocked into the endogenous CTLA4 locus with an age of 6-11 weeks at the day of tumor inoculation. This model is also known as a Human Genetically Engineered Mouse Model or HuGEMM. Mice were subcutaneously implanted with 1×10.sup.6 MC38 tumor cells in the right flank. When tumors to be injected were grown to a mean tumor volume of ˜65 mm.sup.3, mice were randomized into treatment cohorts (day 0) and treated with either: 1) a single 50 μL intratumoral injection of control hydrogel 30 or 2) four total 200 μL intraperitoneal doses of 18 μg given on days 0, 4, 8, and 12 of free CTLA-4 mAB, or 3) a single 50 μL intratumoral injection of 840 μg of CTLA-4 mAB-linker-hydrogel prodrug 29b or 3) a single 50 μL intratumoral injection of 72 μg of CTLA-4 mAB-linker-hydrogel prodrug 29c. Hydrogels were administered as suspensions in 20 mM succinate, 135 mM NaCl, 0.01% Tween20, pH 4.0 buffer.

[1516] Mice were sacrificed 7 days after randomization (D0). Following sacrifice, blood, spleen and tumor were isolated. Spleen samples were dissociated mechanically to generate single suspensions. Tumor samples were enzymatically and mechanically dissociated to generate single cell suspensions. Cell suspensions from spleen and tumor were centrifuged at 300 g for 5 minutes and 2×10.sup.6 cells were used for staining. For whole blood, approximately 100 μL was used for staining. Cells were resuspended in FACS buffer with 1 μg/ml Fc-Block and incubated at 4° C. for 10 minutes in the dark. Surface marker antibody mixtures in FACS buffer were added to each sample and samples were incubated in the dark at 4° C. for 30 minutes. Red blood cell lysis buffer (Bio-gems) was added if needed and cells were further incubated at 4° C. for 10 minutes. Cells were washed twice with FACS buffer then fixed and permeabilized for 30 minutes at room temperature with Fix/Perm buffer (eBioscience). Cells were washed twice in Permeabilization Buffer and stained with intracellular antibodies in Permeabilization buffer for 30 minutes at room temperature. Cells were washed twice in FACS buffer and acquired in the presence of 123count Ebeads (eBioscience). After collection, FACS data was analyzed using FlowJo Version 10.6.1. Compensation was digitally adjusted using single antibody-stained beads, single antibody-stained cells, and fluorescence minus one (FMO) controls. CD4.sup.+ T cells were gated as live, intact CD45+ CD3+ CD4+ events. Tregs were gated as the CD25+ FOXP3+ subset of CD4 T cells. CD8.sup.+ T cells were gated as live, intact CD45+ CD3+ CD8+ events. Subgates were then defined using the controls mentioned above. The change in the mean percentage of positive cells for indicated populations relative to control treatment was calculated by the formula: mean percentage of positive cells in treated animals—mean percentage of positive cells in control treated animals.

[1517] Summary of Antibodies Used for FACS Profiling:

TABLE-US-00008 Markers Fluorochrome Clone Isotype CD45 FITC 30-F11 Rat IgG2b, κ CD3 BUV395 17A2 Rat IgG2b, κ CD4 BUV737 GK1.5 Rat IgG2b, κ CD8 PE-eFluor610 53-6.7 Rat IgG2a, k CD25 BV510 PC61 Rat IgG1, λ FOXP3 PE FJK-16S Rat IgG2a, k Ki67 PerCP/Cy 5.5 16A8 Rat IgG2a, k ICOS BV421 C398.4A Armenian Hamster IgG CD69 BV605 H1.2F3 Armenian Hamster IgG CD44 BV711 IM7 Rat IgG2b, κ CD62L APC MEL-14 Rat IgG2a, k CD335 PE/CY7 29A1.4 Rat IgG2a, k Live/Dead efluo780 NA NA

[1518] Results:

TABLE-US-00009 TABLE 5 Increase in Percentage Positive from control hydrogel 30 Tissue + Readout Tumor Tumor Tumor Tumor Treatment CD44++ CD44++ CD62L− CD69+ ICOS+ CD62L− of CD4s of CD4s of CD4s of CD8s CTLA-4 mAB 10.73% 2.37% 13.77% 6.51% 29b 34.33% 6.85% 18.12% 48.45% 29c 40.55% 13.54% 11.42% 44.73%

TABLE-US-00010 TABLE 6 Increase in Percentage Positive from control hydrogel 30 Tissue + Readout Blood Blood Blood Blood Blood Blood Treatment CD4 Treg CD25+ CD69+ CD69+ ICOS+ of T of CD4 of CD4 of CD4 of Treg of CD8s CTLA-4 mAB 0.53% 2.22% 2.88% 3.35% 3.69% 9.26% 29b −11.03% 2.58% 6.58% 3.83% 3.21% 5.86% 29c −0.43% 1.20% 2.09% 1.47% 2.77% 1.86%

TABLE-US-00011 TABLE 7 Increase in Percentage Positive from control hydrogel 30 Tissue + Readout Spleen Spleen Spleen Spleen Spleen Spleen Treatment CD4 Treg CD25+ CD69+ CD69+ ICOS+ of T of CD4 of CD4 of CD4 of Treg of CD8s CTLA-4 mAB 4.90% 6.64% 4.38% 8.33% 9.17% 3.18% 29b −2.10% 5.84% 4.25% 7.33% 9.87% 0.65% 29c −0.83% 1.57% 1.49% 2.87% 4.73% 0.17%

TABLE-US-00012 TABLE 8 Increase in Percentage Positive from control hydrogel 30 Tissue + Readout Blood Blood Blood Spleen Spleen Treatment CD44++ CD62L− ICOS+ Ki67+ Ki67+ Ki67+ of CD4s of CD4s of CD4s of CD4s of Tregs CTLA-4 mAB 15.28% 18.80% 14.64% 11.75% 16.97% 29b 8.51% 9.04% 8.28% 3.82% 5.40% 29c 3.60% 2.27% 2.32% 0.36% −0.15%

[1519] In an analysis of intratumoral lymphocytes for measures of local T cell activation, treatment with 29b or 29c resulted in increased local T cell activation in tumors as compared to 30 (Table 5). For example, as compared to 30, treatment with 29b or 29c resulted in increases in the percentage of CD44.sup.++CD62L.sup.1ow effector cells within CD4 T cells (34.33% and 40.55%, respectively); increases in the percentage of CD69.sup.+ cells within CD4 T cells (6.85% and 13.54%, respectively); increases in the percentage of ICOS.sup.+ cells within CD4 T cells (18.12% and 11.42%, respectively); and increases in the percentage of CD44.sup.++CD62L.sup.1ow effector cells within CD8 T cells (48.45% and 44.73%, respectively).

[1520] In an analysis of peripheral blood or splenic lymphocytes as measures of systemic anti-CTLA4 induced responses, treatment with 29b or 29c resulted in limited systemic CTLA4 induced responses as compared to 30 (Tables 6, 7, 8). For example, compared to 30, treatment with 29b or 29c resulted, in peripheral blood (Table 6), in no increases in the percentages of CD4 T cells within total T cells and less than 10% increases in: the percentages of T.sub.regs within CD4 T cells (increases of 2.58% and 1.20%, respectively); the percentage of CD25.sup.+ T cells within CD4 T cells (increases of 6.58% and 2.09%, respectively); the percentage of CD69.sup.+ T cells within CD4 T cells (increases of 3.83% and 1.47%, respectively); the percentage of CD69.sup.+ cells within T.sub.regs. (increases of 3.21% and 2.77%, respectively); or the percentage of ICOS.sup.+ cells within CD8 T cells (increases of 5.86% and 1.86%, respectively). Similarly, compared to 30, treatment with 29b or 29c resulted, in spleen (Table 7), in no increases in the percentages of CD4 T cells within total T cells and less than 10% increases in: the percentages of T.sub.regs within CD4 T cells (increases of 5.84% and 1.57%, respectively); the percentage of CD25.sup.+ T cells within CD4 T cells (increases of 4.25% and 1.49%, respectively); the percentage of CD69.sup.+ T cells within CD4 T cells (increases of 7.33% and 2.87%, respectively); the percentage of CD69.sup.+ cells within T.sub.regs. (increases of 9.87% and 4.73%, respectively); or the percentage of ICOS.sup.+ cells within CD8 T cells (increases of 0.65% and 0.17%, respectively).

[1521] In an analysis of peripheral blood or splenic lymphocytes as measures of systemic anti-CTLA4 induced responses, as compared to 30, treatment with 29b or 29c resulted in limited systemic CTLA4 induced responses as compared to treatment with CTLA-4 mAB (Table 8). This is noteworthy as both CTLA-4 mAB and 29b resulted in similar efficacy of anti-tumor growth inhibition (Table 4). Uniquely, as compared to treatment with 30, treatment with 29b or 29c but not CTLA-4 mAB resulted in less than 10% increases in: the percentage of CD44.sup.++CD62L.sup.1ow effector cells within CD4 T cells in blood (increases of only 8.51% and 3.60%, respectively); the percentage of ICOS.sup.+ cells within CD4 T cells in blood (increases of only 9.04% and 2.27%, respectively), the percentage of Ki67.sup.+ cells within CD4 T cells in blood (increases of only 8.28% and 2.32%, respectively), the percentage of Ki67.sup.+ cells within CD4 T cells in the spleen (increases of only 3.82% and 0.36%, respectively), the percentage of Ki67.sup.+ cells within T.sub.regs in spleen (an increase of only 5.40% and a decrease of 0.15%, respectively). The lack of induction of these systemic activation markers with water-insoluble controlled-release anti-CLTA4 compound of the present invention at a dose demonstrating TGI as demonstrated here is significant and noteworthy as these markers are typically induced by systemic anti-CTLA4 therapy which is known to be associated with systemic adverse events.

Abbreviation

[1522] AcOH Acetic acid [1523] Asp aspartate [1524] Boc tert-butyloxycarbonyl [1525] DCC N,N′-Dicyclohexylcarbodiimide [1526] DCM Dichloromethane [1527] DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene [1528] DCU N,N′-Dicyclohexylurea [1529] DIC N,N′-diisopropylcarbodiimide [1530] DIPEA N,N-Diisopropylethylamine [1531] DMAP 4-(Dimethylamino)-pyridine [1532] DMF N,N-Dimethylformamide [1533] DMSO Dimethyl sulfoxide [1534] DS Degree of substitution [1535] EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide HCl salt [1536] EDTA Ethylenediaminetetraacetic acid [1537] EtOAc Ethyl acetate [1538] Fmoc Fluorenylmethyloxycarbonyl [1539] HA Hyaluronic acid [1540] HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid [1541] HOBt 1-Hydroxybenzotriazole [1542] HOSu N-Hydroxysuccinimide [1543] HPLC High-performance liquid chromatography [1544] IAA iodoacetamide [1545] LC Liquid chromatography [1546] LC-MS Mass spectrometer coupled liquid chromatography [1547] LPLC low pressure liquid chromatography [1548] MES 4-Morpholineethanesulfonic acid [1549] MTBE Methyl tert-butyl ether [1550] MTS Methanethiosulfonyl [1551] Mw Molecular weight [1552] NHS N-Hydroxysuccinimide [1553] NMP N-Methyl-2-pyrrolidone [1554] OPA o-Phthalaldehyde [1555] PE Polyethylene [1556] PEG Polyethylene glycol [1557] PES Polyethersulfone [1558] PTFE Polytetrafluoroethylene [1559] PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphat [1560] r.t./rt Room temperature [1561] RP reversed phase [1562] RP-HPLC Reversed-phase high-performance liquid chromatography [1563] SPDP N-Succinimidyl 3-(2-pyridyldithio)propionate [1564] tBu and t-Bu tert.-butyl [1565] TCEP Tris(2-carboxyethyl)phosphine hydrochloride [1566] TFA Trifluoroacetic acid [1567] THF tetrahydrofurane [1568] TMEDA Tetramethylethylenediamine [1569] TSTU N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uroniumtetrafluorborate [1570] UPLC Ultra performance liquid chromatography