Granted Patent B2

US 12,239,738 · App. 17/503,647 · Granted Mar 4, 2025

Inventors: David Fela (Califon, NJ); Anu Wallecha (Yardley, PA); Mike Grace (Monmouth Junction, NJ); Melissa Gosse (Jackson, NJ)

Assignee: Advaxis, Inc.

A61K9/19A61K35/74C12N1/20

Abstract

Claims (35)

1. A formulation for lyophilization of a Listeria strain, comprising: (1) the Listeria strain; (2) a phosphate buffer; and (3) sucrose,

wherein the Listeria strain is a recombinant Listeria monocytogenes strain,

wherein the formulation comprises 1% to 5% w/v sucrose, and

wherein the formulation does not comprise trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA).

2. The formulation of claim 1 , wherein the Listeria strain is a strain in which a stress response has been induced by exposing the Listeria strain to a decreased temperature.

3. The formulation of claim 1 , wherein the Listeria strain is from a frozen Listeria stock.

4. The formulation of claim 1 , wherein the Listeria strain is from a freshly cultured Listeria stock.

5. The formulation of claim 1 , wherein the formulation comprises about 2% to about 3% w/v sucrose.

6. The formulation of claim 5 , wherein the formulation comprises about 2.5% w/v sucrose.

7. The formulation of claim 1 , wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide.

8. The formulation of claim 7 , wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in prfA, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

9. The formulation of claim 7 , wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

10. A lyophilized composition comprising a Listeria strain produced by a method comprising:

(a) providing a composition comprising a Listeria strain in a formulation comprising a buffer and sucrose,

wherein the Listeria strain is a recombinant Listeria monocytogenes strain,

wherein the formulation comprises 1% to 5% w/v sucrose, and

wherein the formulation does not comprise trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA);

(b) cooling the composition provided in step (a) at a holding temperature between about −32° C. and about −80° C. in a freezing step;

(c) exposing the composition produced by step (b) to a vacuum at a holding temperature between about −10° C. and about −30° C. in a primary drying step; and

(d) exposing the composition produced by step (c) to a vacuum at a holding temperature between about −5° C. and about 25° C. in a secondary drying step,

whereby the lyophilized composition is produced, and

wherein the residual moisture in the lyophilized composition is between about 1% and about 5%.

11. A lyophilized composition comprising a Listeria strain, a phosphate buffer, and sucrose,

wherein the Listeria strain is a recombinant Listeria monocytogenes strain,

wherein the composition does not comprise trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA), and

wherein the residual moisture in the lyophilized composition is between about 1% and about 5%.

12. The lyophilized composition of claim 11 , wherein the residual moisture in the lyophilized composition is between about 2% and about 4%.

13. The lyophilized composition of claim 11 , wherein the residual moisture in the lyophilized composition is at least about 2.5%.

14. The lyophilized composition of claim 13 , wherein the residual moisture in the lyophilized composition is at least about 3%.

15. The lyophilized composition of claim 11 , wherein the lyophilized composition shows at least about 60% viability after storage at between about −20° C. and about 4° C. for about 12 months.

16. The lyophilized composition of claim 15 , wherein the lyophilized composition shows at least about 75% viability after storage at between about −20° C. and about 4° C. for about 12 months.

17. The lyophilized composition of claim 16 , wherein the lyophilized composition shows at least about 80% viability after storage at between about −20° C. and about 4° C. for about 12 months.

18. The lyophilized composition of claim 11 , wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide.

19. The lyophilized composition of claim 18 , wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in prfA, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

20. The lyophilized composition of claim 18 , wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

Assignments (2)

Continuity (3)

References Cited (257)