# US 8,541,551 B2 · Immunogen and antivenom against violin spider venom

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- Number: US 8,541,551 B2
- Document ID: US8541551B2
- Status: Granted
- Kind: B2
- Application number: 13/610,516
- Filed: 2012-09-11
- Published: 2013-03-21
- Granted: 2013-09-24
- Earliest claimed: 2004-08-31
- Assignees: Universidad Nacional Autonoma De Mexico; Laboratorios Silanes S.A. de C.V.
- Applicants: Alejandro Olvera Rodriguez; Roberto Pablo Stock Silberman; Blanca Margarita Ramos Cerrillo; Rosana Sanchez-Lopez; Alejandro Alagon Cano
- Inventors: Alejandro Olvera Rodriguez; Roberto Pablo Stock Silberman; Blanca Margarita Ramos Cerrillo; Rosana Sanchez-Lopez; Alejandro Alagon Cano
- Web page: https://patentgrep.com/patent/US8541551B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- A61P 37/04: Immunostimulants
- A61P 39/00: General protective or antinoxious agents
- A61P 39/02: Antidotes
- C07K 14/43518: {from spiders}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention relates to the isolation, characterization and expression of DNA fragments encoding sphingomyelinases D from three species of Loxosceles genus spiders, namely L. boneti., L. reclusa and L. laeta, and the toxoids thereof. The invention also relates to the production of active sphingomyelinases D and the toxoids thereof using recombinant means and to the use of same as an immunogen for the production in vertebrates of antibodies that neutralize the corresponding venom and the respective fragments F(ab′)2. The invention further relates to the use of recombinant sphingomyelinases D as part of an antigen matrix which can be used in the immunopurification of antibodies and the fragments thereof or as part of any diagnostic device used to obtain clinical confirmation that the causal agent of poisoning in a patient is a spider of the Loxosceles genus. In addition, the invention includes molecular vectors for the expression of the DNA fragments, strains comprising same, which can express Loxosceles sphingomyelinases D, and methods for the express thereof.

## Claims

### Claim 1

A recombinant toxoid selected from the group consisting of SMDrLb1(H11K) and SMDrLb1(E31K), wherein the toxoid has an amino acid sequence as provided in SEQ ID NO: 28 and SEQ ID NO: 30, respectively, the toxoid characterized by being an antigen lacking sphingomyelinase D property, by not being dermonectrotic, and by being able to generate neutralizing antibodies against the venom of Loxosceles boneti.

### Claim 2

A method of neutralizing the venom of Loxosceles boneti or for treating the poisoning caused by Loxosceles boneti comprising administering a recombinant toxoid selected from the group consisting of SMDrLb1(H11K) and SMDrLb1(E31K) to a patient in need thereof, wherein the toxoid has an amino acid sequence as provided in SEQ ID NO: 28 and SEQ ID NO: 30, respectively, the toxoid characterized by being an antigen lacking sphingomyelinase D property, by not being dermonectrotic, and by being able to generate neutralizing antibodies against the venom of Loxosceles boneti.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the recombinant toxoid optionally include a histidine tail at the C or N terminus region.

### Claim 4

A method for obtaining an antivenom to neutralize the venom of Loxosceles boneti spiders comprising:
a. immunizing a non-human, vertebrate animal with an antigen selected from the group consisting of:
i. SMDrLb1 (H11K), SEQ ID NO: 28;
ii. SMDrLb1(E31K), SEQ ID NO: 30 and
iii. a mixture thereof
b. obtaining serum or plasma from the animal and isolating the polyclonal antibody fraction;
c. optionally hydrolyzing the polyclonal antibody fraction to obtain an F(ab)2 fragment of the polyclonal antibody;
d. determining the DE50 of the polyclonal antibody or the F(ab)2 for neutralizing the venom of Loxosceles boneti spiders; and
e. obtaining an effective dose based on the determined DE50.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the vertebrate is a mammal.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the vertebrate is an equine.